Critical Reviews in Biotechnology, 13(3):195-253 (1993)

Problems in Scale-Up of Biotechnology

Production Processes
Harold B. Reisman
Organogenesis Inc., 150 Dan Road, Canton, MA 02021

ABSTRACT: The unique nature of biotechnology processes adds to the complexity and difficulty of scale-up.
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Successful scale-up means a shortened cycle to full-scale production, competitive advantage, and cost savings. The
many pitfalls as well as actual and potential scale-up problems are reviewed. Emphasis is placed on covering all
areas of concern in planning, executing, and documenting key studies. Needs in technology transfer are discussed
and regulatory requirements are incorporated into scale-up needs. A review of the recent literature is coupled with
actual case studies; problem avoidance is stressed. Problems in asepsis, in construction, and in validation are
discussed and potential solutions given. Organizational problems are noted. Finally, checklists are given for project
planning, for a safety audit, and for timely attainment of successful scale-up. Eighty-two references are included.

KEY WORDS: bioreactor, agitation-aeration, sterilization, asepsis, validation, cell culture, fermentation, project
planning, downstream processing, containment.
For personal use only.

I. INTRODUCTION 4. Illness or discomfort to consumers

5. Argumentation and abuse for the perpetrator
It will never be known with certainty which
fermentation process occurred first in human his- Therefore, the first fermentation process was
tory. There is ample evidence that beer, bread, quickly followed by the first scale-up problem in
and wine were produced in quantity in ancient a biotechnology process. Interestingly, the results
Egypt - that is, over 6000 years ago. The likeli- previously noted are not uncommon when a con-
hood is that a food fermentation occurred acci- temporary scale-up problem ensues. Of course,
dently and the resulting product was more appre- today we can add one or more of such factors as
ciated than the starting material. The fermentation monetary loss, loss of business credibility, and
could have occurred in a dairy product, in an early undesirable regulatory and legal consequences.
kind of bread, or in an alcoholic ferment. Very Scale-up and problems seem to go together.
likely, preservation and/or taste were enhanced. This has been the norm. After reviewing the
Because the mechanism was unknown, we can many real and potential problems of scale-up,
presume that one of our progenitors tried - in a one might legitimately assume that no biologic
purposeful way -to simulate that early fermen- process can be scaled up. Fortunately for mod-
tation process. It is likely, but by no means cer- ern man, this is not the case. Long lists of useful
tain, that early attempts resulted in one or more of fermentation products -made in bulk -can be
the following: generated. We can list amino acids, antibiotics,
vitamins, biopolymers, monoclonals, vaccines,
1. Loss of a desirable starting material (sub- enzymes, and physiologic proteins with dollar
strate) values in the many billions of dollars annually.
2. Creation of a large volume of a useless Still, the path from research and discovery to
mixture large-volume production has rarely been a direct
3. Foul odor or a foamy mess and easy one.

0738-855 1/93/$.50
0 1993 by CRC Press, Inc.
195
 In this review, 1 do not separate the “older” predicted and no unexpected entities are found in
fermentation processes (commodities, bacterial, the product. Although optimization may increase
fungal, yeast) and their inherent scale-up prob- yield and throughput before inauguration of large
lems from the “newer cell culture processes.’’ scale production, it is hazardous to “design in”
Although there are unique differences and unique process improvement.
solutions (and some will be discussed), there are If successful scale-up results in timely pro-
many more similarities in the scale-up method, in duction of the desired quantity of product, an-
recognition of potential pitfalls, and in the plan- other result is (hopefully) a process that can be
ning of the successful scale-up effort. There are validated at production scale. Clinical quality and
many issues common to the various biotechnol- quantity are achieved and appropriate documen-
ogy processes and even if emphasis and approach tation is available for regulatory purposes.
must vary, the overview concept might stimulate The short answer to the question of scale-up
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an insight that compartmentalization would tend importance consists of two words: time and money.
to stifle. Most emerging companies are short of both com-
Scale-up refers to any increase according to a modities. Any delay or problem in scale-up means
fixed ratio. Strictly, therefore, transfer of a pro- loss of one and probably both needs. The longer
cess from a shake flask to a 5-1 fermentor is a answer to the question involves theoretical and
scale-up step. This level of scale-up is not free empirical understanding of
from hazard, but the subject matter to follow
stresses scale-up where the fixed ratio may be 1. Transport phenomena in animal, plant, in-
2000 or more. The scale-up problems reviewed sect cells at various scales with various sup-
occur in the transfer of a research project or pro- port media
cess through development into volume manufac- 2. Shear impact on these cells in mechanically
For personal use only.

turing, It should seem obvious to all concerned and/or gas-mixed systems

that scale-up is a serious matter and involves 3. Genetic stability
careful planning and careful execution. The fact 4. Materials of construction (both synthesis and
that scale-up is seldom simple indicates that the isolation)
obviousness is not clear to everyone who should 5. Isolation methods that are currently in lab or
know better. There is no other explanation for the small scale use and novel purification meth-
many repeated problems that arise in the course ods
of scale-up. Enough is known about scale-up to 6. Compound molecular configuration (tertiary
recognize the potential for probIems and, at the characteristics as relates to stability and
minimum, to plan for problem-avoidance. Why is potency)
successful scale-up so important? 7. Control strategies for effective biosynthesis
One major requirement for commercial suc- and isolation
cess is investment capital. A government publica-
tion’ has a terse summary that answers part of the Successful scale-up means that a number of
question note above: issues in these various areas have been faced and
resolved. Even if complete understanding is not
Several factors have been cited for tightened avail- achieved, a background for future development
ability of venture capital financing. Product develop- has been established. Successful scale-up is nec-
ment was slower than expected (e.g.. unforeseen tech-
nical problems, slow regulatory approval and patent
essary (but not sufficient) for either production
issuance, and difficulties in Scale-Up and in obtaining and widespread use of bioactive agent or com-
meaningful clinical results). mercial success. Often, successful scale-up pre-
cedes both achievements.
Successful scale-up, almost by definition, Why is scale-up an ongoing problem? To be
means that a process has been designed and built simplistic, every process involving living organ-
giving a predictable increase in production capac- isms is somewhat unique. The organism, often
ity. It is understood that product potency is as highly selected and/or modified, interacts and

196
 alters its environment in a very complex manner. however, these parameters, among others, will
Changes in the environment -caused by a change change: bulk mixing/turnover time, cost (both
in scale - will cause unpredictable responses by initial capital investment and operating), foaming
the organism that will further modify the environ- characteristics, shear, pCO,, gas-liquid interfacial
ment at the next scale. Very often, these interac- area. An understanding of acceptable limits will
tions cause unexpected results that are usually focus the scale-up effort and result in the most
economically unattractive. At times, results are cost-effective and easiest-to-operate production
economically disastrous. facility.
It is probably a truism to state that no com- In an overall review of scale-up, it is useful to
plete set of chemical/physical conditions imposed have some grasp of the qualitative aspects of
at a manufacturing scale will result in the same microbial systems involved. Although no single
environment that existed in shake flasks or table can cover the spectrum of microbial sys-
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T-flasks. Therefore, an early and obvious premise tems, general concepts can be given as a guide to
is that numerous compromises must be made in scale-up considerations. As can be seen in Table 1,
the course of scale-up. Success or failure of scale- there is no overall rule or plan for scale-up that is
up depends on how close rate, yield, and purity at applicable across the board. In general, pure bac-
the larger scale matches those results at the bench. terial and fungal systems (excluding recombinant
Because it is not possible to keep all chemical/ cells for the moment) have been in use for many
physical parameters constant on changing scale, decades. Sterility control, inoculum development,
it is important to know what boundaries exist for strain improvement, and control strategies are
successful implementation. As one example - fairly well understood. Successful scale-up has
there are combinations of agitation and aeration occurred. These aspects of biotechnology devel-
that will give an equivalent result. As changes are opment and scale-up are less clear in the area of
For personal use only.

made in one or both of these input variables, animal and plant cell biosynthesis. Further, novelty

TABLE 1
Characteristics of Microbial and Cell Culture Systems
Bacteria Moldlfungi AnimaVplant cells

Doubling time Shortest Intermediate Slowest (days)

Viscosity Normally low Often a problem Low
Medium cost Low Low to High
intermediate
Shear resistance High Often high Often low
Cost of downstream Modest Modest High
processing (5050)” (10:90)”
Culturing Suspension Suspension Substrate or suspension
Product concentration High Intermediate Can be very low
Aggregation Nil to low Low to May be a problem
intermediate
Product value Low to Low to high High to very high
intermediate
Cell density Can be very high Intermediate to Usually low
high
Clean steamMlFl Almost never Almost never Very often
Contamination Nil to low Nil to low Often a problem
Genetic stability Usually stableb Occasional Sometimes a problem
problemsb

a Ratio of Synthesis:lsolation cost as percentages (total = 100°/o).

If genetically modified, plasmid stability is often a problem.

197
 in both synthesis and isolation require more care- disregarding the potential for difficulty; such an
ful regulatory oversight. Design requirements and approach will lead to difficulty at some point.
problems in process implementation flow from Furthermore, if knowledge or experience is not
the qualitative characteristics presented. It will be available in one research or development group, it
seen that the problems and solutions relate quite should be sought elsewhere, inside or outside the
strongly to qualitative factors; this should not be company. To select some examples:
surprising. In fact, the sensitivity of animal and
plant cells to media constituents, to agitation- 1. Sterility at the lab bench is not the same as
aeration conditions, and to contamination means sterility in a production environment. Steril-
that more lab scale experimentation must be per- ity control is often considered “someone
formed and that any slight oversight in design else’s problem.” Operating in a laminar flow
will result in a scale-up problem. hood is not the same as operating in a Class
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The qualitative review leads to a listing of 10 room. Some sterile techniques are emi-
scale-up problems. Not every issue shown nently suitable for small volumes; the same
(Table 2) occurs in every scale-up situation. Every technique may be very difficult or impos-
issue has the potential to arise at one or another sible on a large scale. It must be recognized
stage of the scale-up process, however, and in the that a nonvalidated change in process (on
author’s experience, each item has arisen in dif- scale-up) may be acceptable from the view
ferent biologic processes. Unfortunately, the list of asepsis and be totally unacceptable for
is not exhaustive and new problems (process spe- process response.
cific) arise for a new product or process. It is best 2. Sterilizing a small volume (say 1 1 or less),
to give some attention and consideration to each in an autoclave is Rot the same as sterilizing
item (including subparts) shown. Assuming “no 5000 1 (or even 400 kl), especially where
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problem” in a specific area is almost the same as heat-labile substances are present. Total heat

TABLE 2
Scale-Up Problems

Asepsis Containment

Volume of inputs Aerobic

media flanges
water connectors
air, other gases pressurization
neutralizing agents gas compressors (additives)
Sterilization effluent inactivation

Time Cleaning

Heating/cooling cycles Fouling

Holding time (stability) Scale formation
Transfer time Equipment reuse
Lag phases Equipment inspection
Agents to use (and disposal)

Volume (Vessel) Regulatory

Turnover time (homogeneity) 10, OQ, PQ (Validation)

Heat transfer (heat load) cGMP (Code of Federal Regulations)
FilVemptying time Waste disposal
Internal fittings Local codes (especially rDNA)
Finish
Material of construction

198
 input varies as does the opportunity for media there may be a severely detrimental ef-
modification. fect. The time response must be verified.
3. Containment requirements are never easy, Hold times may be minutes in the lab;
and more stringent requirements may exist they may be hours in a plant. Shelf life
in a laboratory than are strictly needed. The and stability are time-related variables.
same high level of containment in a pro- Prediction is difficult and may be hazard-
duction facility may be onerous and costly ous to the process.
. . . and unnecessary. Containment need 5. Cleaning often receives cursory attention.
should be jointly determined with appro- Scale formation in a small glass vessel is
priately trained personnel. solved by buying a new vessel. Hand wash-
4. The concept of “time” does not receive ap- ing is feasible in the lab. Equipment inspec-
propriate attention. Time increments that are tion is relatively easy in a lab. All these
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reasonable and meaningful in the laboratory aspects of cleaning are rendered more com-
(such as “lo min in trypsin at 35°C”) cannot plex or more difficult in a production envi-
be scaled up indefinitely. Transfer of inocu- ronment. Gaseous or liquid residues in an
lum may take less than 30 sec in the lab; this incubator or in bioreactors may result from
may translate to 30 min at production scale. commercially available, but not thoroughly
There may be no impact on the inoculum or tested, cleaning/sanitizing agents. Many

TABLE 3
Scale-Up Considerations (Potential Concerns)
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Potential Concerns
~~

Strain selection Purity Aggregation

Stability Viscosity
Mutation Degeneration
Byproducts Phage resistance
Raw materials Purity (vendor audit) Availability
Standards Cost
Uniformity Substrate concentration
Interactions Defoamer
lnoculum development Transfer time Storage parameters
Hold time Number of vessels
Number of generations Quality control
Sterilization Total heat input Maillard reaction
Method used Hold time
Fouling
Selection of parameters Mixing (turnover) Power and shear
DO, level CO, level
Homogeneity Oxygen transfer rate
PH ( O W Pressure
Gradients Temperature
Materials of construction and cleaning Ionic contaminants Surfactants
Corrosionlerosion Agents used
Release agents Surface and weld quality
Monitoring Sensor stability Quality of control
Response time Alarms (and response)
Sampling
Harvest Transfer time Cell degradation
Hold time (stability) Asepsis
Isolation Stability Impurities
Cost (quality of control) Recycle streams
Regeneration

199
 cleaning and sanitizing agents require not normally do not elicit concern when a superior
only special handling but special disposal mutant is introduced, insertion of plasmids may
techniques. elicit concern.
A second example concerns a superior pro-
A possible scenario for these potential ducer (at culture flask level) that - for some
problems as well as others listed would be to reason -does not give enhanced productivity at
raise each issue at an early joint meeting in- plant scale. What is optimal for culture 101 is
volving research, development, and manufac- very often suboptimal for culture 125, even
turing personnel. Each area of uncertainty though the latter always outperforms the former
should be assessed and any further effort (ac- in small-scale tests. It is not uncommon for the
tual research work or a literature review, for test culture to perform below its “control (lab)”
example) defined. Minutes should be kept and level at the production scale. It requires skill,
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a clear historical line maintained that reviews patience, and attention to detail to pursue paral-
solutions or dispositions of each potential prob- lel paths. What changes should be investigated
lem. on scale-up to mirror improved productivity?
The same sort of scenario can be extended to How long should one work on a “superior” cul-
a more complete listing of items that require re- ture before proceeding to the next mutant cul-
view; here, theoretical and practical considerations ture? Other questions to consider are: Does the
(with some detail) can be shown. In Table 3, new culture require enhanced mixing? Does the
scale-up considerations are shown; the format is new culture require a modified medium or a
more of a checklist. Not every consideration will modified feeding schedule? What is sensitivity
result in a scale-up problem; some points are not of the new mutant to changes in DO level or in
relevant in certain circumstances. However, as- response to different CO, tension? Is the new
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suming “no problem” with insufficient informa- mutant more sensitive to variation in ion content
tion or input is definitely the wrong choice. A or concentration?
clear statement (preferably supported by experi- The balance of effort between the research
mental evidence) in the technology transfer docu- lab, the development group, and manufacturing
ment concerning each of the issues will pay off is not a simple matter, as these few examples
handsomely in subsequent scale-up and timely show.
accomplishment.
Another difficulty in scale-up is rather com-
mon. It is extraordinarily difficult to integrate II. PROJECT PLANNING
strain development and process development. Two
examples follow. Later in this review, selected organizational
Assume media optimization is ongoing as requirements are discussed. For a project such as
scale-up proceeds. Adjustment up or down for a scale-up to be implemented successfully (or to
constituent listed in SOPS and documentation avoid serious problems), it is important to under-
available for later regulatory review presents no stand something about project planning and project
problem (in most cases). If a new ingredient is implementation. To avoid scale-up problems, at
added or one is completely deleted and product least these elements should be put in place:
yield or rate is enhanced, however, a problem
may or may not arise. Are any new byproducts Establish a multidisciplinary team
introduced? Is product purity unchanged? Is sta- Formally or informally, select or acknowledge
bility impacted? There are other questions, many a product or process champion
of which are related to regulatory oversight. It is Require early involvement by all
clear that any genetic alternation in the producing Develop scope of work, design criteria, and
organism must be reviewed with great care. Al- definition of success (goals/objectives)
though generalized mutation programs (antibiotic Establish some flexibility or alternatives in
biosynthesis, amino acid formation, as examples) planning

200
 Establish a budget (or questions to be answered different. In general, however, a scale-up project
so that a budget can be set in place) will involve some amount of design or engi-
Establish means of communication, both for- neering, some amount of equipment/materials
mal and informal procurement, some amount of construction/
modification, and some time for start up or
For a more formal planning checklist see demonstration. Even the simplest project re-
Table 4.This article will not give every detail quires some attention to:
that must be checked in the course of a scale-up
project. A scale-up project may be as “simple” Process flow diagram (PFD)
as running a new mutant in existing equipment Equipment list (existing, modified, or new)
with no new construction or as complex as build- Energy, utility requirements
ing a new product facility with labs and offices Raw materials list and usage
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needed for support functions. Obviously, both Equipment layout (including personnel flow)
time required and funds needed will be vastly Regulatory impact (including health and safety)

TABLE 4
Project Planning Checklist
(Scale-Up Program)

Objectives
Scope of work (answer what? when? how much?)
Location of facility (new, expansion, or retrofit)
Schematic process flow diagram (PFD)
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Need for architect? engineering consultants?

Environmental impact study
Regulatory issues (product, process, environment, personnel)
Alternatives (value engineering)
Time for construction
Time for break-in or start up
Time for demonstration
Budget and accounting procedures
Internal communications, meeting schedule, reporting
With all required inputs, develop a bid package (or request for proposal, abbreviated
RFP)
Check on time schedule (after receipt of delivery/constructiontimes)
Readjust or reevaluate Items 1 and 2 in light of responses
Develop a final schedule
Consider regulatory review of plans (consultant or government)
Identify long delivery items and place orders
Identify special utilities requirements and schedule/emergency power
Auxiliary services or inputs to program (if not included above):
Engineering Quality Control
Regulatory Quality Assurance
MIS Health and Safety
Establish contractual relationship with contractors
Refine final schedule and budget
Establish construction oversight and initiate construction
Establish start-up team and start-up schedulelSOP preparation
Building permits, specialty alarms (prior to construction)
Establish document receiptldocument control function
Establish validation schedule
Reviewhefine insurance requirements
Establish inspection and testing sequence during start up
Photography (videotape) of construction and start up
Receipt of as-built drawings

201
 If there is any degree of complexity (or needed optimum installation. The completion of con-
investment), provision must be made for: struction requires the same level of care and
vigilance. Contractors are invariably anxious to
Piping and instrumentation diagram (PID) depart once a system is in place. The transfer of
Process equipment specifications responsibility is a key event; many hidden scale-
Instrument and controls package up problems exist at this point in time. It is
critically important to have joint responsibility
Although many scale-up projects are completed and joint oversight at this time (and this applies
successfully without any significant reference to to every key piece of equipment as well as to the
these many details, one should be cognizant of the fully functional facility). Completion of con-
innumerable details that must be addressed to struction has a number of overlapping phases
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prevent project or process problems. If a researcher and steps:

cannot explain why or how a reactor or column is
to be controlled in the lab, the success of the Detailed warranty information and PM sched-
scale-up project will depend on luck. One func- ule
tion of the planning process is to integrate instru- Operating manuals distributed
ment and control needs into the new facility or As-built drawings presented
new process. Process control is critical in the Tagging valves, signage on pipes
laboratory and is just as important in the pilot or Cleaning of piping and equipment (including
full-scale plant. In the project planning cycle (as passivation)
discussed later), the blanks are filled in and the Testing of pumps, pipelines, valves, fittings
key questions are answered. Required hydrostatic tests (code requirements)
Even though procurement and construction Instrument calibration records presented
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require unique skills, I would urge members of Electrical continuity check (emergency circuits
the scale-up team or task force to remain inti- check)
mately involved during these phases. It is usual Control-point checkout sheets, control ele-
to have research and development (R&D) per- ments’ function
sonnel and operating personnel heavily involved Programmable logic controllers (PLC) check-
during the planning phase and then again during out
commissioning and start up. Construction is left Software printout for all relevant controls
to the “experts,” whether internal to the com- Alarm verification
pany or an outside general contractor. This is Creation of punch lists
nonideal. The scale-up team should be involved Special testing (as clean rooms, hoods, ovens,
at periodic progress meetings, at meetings where autoclaves)
alternatives or change orders are approved, and
at meetings where controls are selected and ap- It is apparent that even this limited list offers a
proved. Members of the scale-up team should host of opportunities for later problems. If regu-
perform periodic inspections of the construc- latory requirements (cGMP) are included, the
tion. Location of a gauge or a manual valve is of potential for problems is enhanced. The scale-up
more importance to the operator of the system team should have sufficient and competent per-
than to the plumbing subcontractor. Insulation sonnel to assure a smooth transfer of responsibil-
on certain lines may protect operating person- ity.
nel; this could be missed in design. Clearance After completion, commissioning occurs.
for maintenance or replacements is sometimes Operating procedures should be in place. Nor-
neglected in design; again, responsible parties mally, water batching occurs. Then all routine
who must “live” with the installation will be sequences (sterilization, inoculation, control func-
more critical and vigorous during reviews and tions, agitation-aeration, transfers, and so forth)
during installation. In sum, the scale-up team are run; any correction or fine-tuning can take
should expend all necessary energy to assure place. Finally, raw materials are charged and the

202
 planned process is initiated. Training should have TABLE 5
begun prior to completion of construction and is Special Testing at
continued and intensified at this point in time. Laboratory or Pilot Plant
With this overview of the project cycle itself, Scale
we can move to detailed analyses of specific equip-
Extreme (Suboptimal) Conditions
ment and operations. Points to consider will be Nutrient levels
stressed with the goal being a scale-up process PH
that is free of delays, upsets, untoward responses, Temperature
and the concurrent disarray and financial loss. Exceed shelf life
Shear
Buffer capacity
COdHCO, levels
111. SPECIAL TESTING
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Defoamer
Holding time
After reviewing all the potential problems, is DO*
it possible to develop a definitive listing of spe- lnoculum
Age (time in cycle)
cial tests that will preclude scale-up difficulties? Age (conditions of storage)
Although the answer is “no,” it is possible to Percent inoculum
generate a list of special tests that should be con- Transfer time
sidered. If we assume there is a workable process Refed
and that the response to key and secondary pro- Reinoculation
Cross-inoculation
cess inputs is known in general terms, special
Media
tests should be performed. To avoid scale-up prob- Alternate raw materials
lems, the special tests should be performed at the Resterilization
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smallest scale that gives predictable performance. Oversterilization

The listing covers areas that are overlooked. (See Sterilization method
Table 5 ) These areas often lead to problems when- Water variation
Protectants
ever “no effect” is assumed. Process responses Culture Device
(cell growth, cell metabolism, product rate, and Materials of construction
yield) should be determined in a rigorous and Surface finishltreatment
quantitative fashion. If trials are done properly, Cleaning/descaling
the actual cause of a production problem might be Environment
Pressure fluctuations
traced more easily, that is, an “unknown” cause
Agitator failure
might be explained by a pattern of responses al- Control failure
ready seen during special testing in the course of Recycle (attachment agent)
scale-up. Knowledge of the laboratory process Distribution piping
should give a very good outline of just how bad Lubricants (utilities)
certain upsets can be, that is, some upsets would Treatment (utilities)
Holding time (isolation)
cause a yield reduction; others would cause a loss
of a batch. Because special testing cannot go on
for years nor can every eventuality be covered,
select those variables and tests that (1) have a age conditions (should the production scale batch
reasonable chance of occurring at large scale and be held up for any reason), reinoculation, plus
(2) have the potential for serious damage. Of potential control problems during inoculation.
special importance are those tests of extremes Remember that inoculation transfer time is not
concerning the inoculum. If a production process the same at lab or production scale. It is some-
is to run for several years, one can be assured that times difficult to justify experiments that search
there will be problems in inoculum development. for suboptimal performance; however, there is
A scale-up team should set up and run abnormal good rationale for this effort and the program
inoculum development procedures; include stor- should include such testing.

203
 IV. BIOREACTOR DESIGN Air lift: a tubular, tower vessel
with gas introduction to
Once an organism is selected or modified, induce circulation and
one is faced with the issue of volume produc- mixing
tion. That is, either the cell mass or a product Draft tube: internal cylinder used to
of cellular activity is desired. Even though direct circulation with or
smaller and smaller quantities of bioactive without mechanical
ingredient are required for physiological ef- agitation; gas introduction
fect, some sort of container or reactor is re- from surface or via bottom
quired. Hence, selection of a fermentor is an sparger
early scale-up need. Whereas the very earliest Recycle: two vessels (equal or
fermentations (almost always mixed cultures) unequal volume) with gas
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occurred in leather pouches or even in a hole introduction to induce flow

in a log or in the ground, advances in recent or external cell separation
decades have moved processes toward a more device with return of
controlled environment. concentrated cell slurry
The design most commonly used (in the Fixed bed: use of supports to retain
past 50 to 100 years) is one form or another of cell mass; liquid (sub-
the stirred tank reactor (STR). The start of strate) flow through or
antibiotic production, corresponding in time across bed for bioconver-
to World War 11, was a great stimulus to use sion
and modification of the STR. Why was the Immobilized cell: use of solid supports (may
STR selected? There are a number of reasons, be porous) for cell growth
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all additive. The vessel and agitator design and adhesion; many
were known entities, a three-phase system (gas, applications in cell culture
solid, liquid) could be handled, the reactor
could be closed, the reactor could be built to Whatever system is to be used, control and
many sizes using different metals, heat trans- measurement of many process variables are of
fer could be conveniently accomplished, critical importance. Gradients are normally unde-
cleanability and sterilization were possible, sirable and broad optima for physiologic vari-
controls could be readily introduced, and last ables are not normally found. In modem biotech-
but not least, the vessel could be easily con- nology, these factors must be considered in
verted, that is, it was a multiuse design. The fermentor design:
STR could also be scaled up. It is, therefore,
Materials of construction Mechanical and
no surprise that this design has not been sup-
other seals
planted, although other bioreactors are in use.
Sterility (asepsis) Controllers/sensors
The STR can be used in continuous fermenta-
Multiple feeds Homogeneity
tion (the abbreviation becomes CSTR),
Aeration (multigas) Turnover time
semibatch or interrupted batch procedures are
possible, recycle opportunities are possible, Agitation (shear impact) Defoamer (additive
or mechanical)
bottom or top drive is possible, baffles or coils
Cleaning Containment
can be placed internally, and operation at an
Flexibility (alternate use) Internal finish
overpressure is simple.
Although a complete review of the fer- A complete listing of desirable characteristics
mentor design is beyond the scope of this re- of pilot fermentors is available; the same list is
view, certain alternative designs should be con- useful for production scale, as
sidered. There may be reasons for selection of A microbial system is complex in ways dif-
a novel design. Alternative fermentor designs ferent from complexities related to chemical reac-
include: tion. In microbial systems, not all reactions are

204
 understood. Pathways are unclear or unknown, ing economic advantage . . . if only a certain reac-
even when the desired product is fully character- tor were used. Presumably, the potential buyer is
ized. An upset to a microbial system may cause aware of such a bias. One report discusses
unexpected (often undesirable) responses. The scalability and comes down firmly on the side of
organism to be grown is often highly selected modularity. In this case, there are some valid
and/or modified; it interacts with and modifies its points to consider, even if some commercialism is
immediate environment in a complex manner. i n ~ o l v e dThe
. ~ most obvious advantage is match-
Changes in the microbial environment - some- ing of production capacity with demand (re-
times caused by a change in scale - will cause validation would probably not be necessary).
unpredictable responses by the organism. These Mobility and compactness are other desirable fea-
responses may further modify the environment at tures. Cleaning and sterilizing modular compo-
the altered scale. Often, the results are benign. On nents will probably be simpler. Inoculum level is
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other occasions, the interactions cause unexpected also “modular”, that is, a number of small inocula
results that are usually economically unattractive. is needed rather than a single large mass. Con-
On occasion, the results are economically disas- tamination of a large batch will clearly be an
trous. Therefore, the scale-up process for the fer- event compared with loss of one of ten modules.
mentor has as its focus the creation of the ideal Capital investment is also modular. The consider-
environment for the cell mass to perform the de- ations are worthy of review prior to making a
sired transformation. selection.
As is true in other areas of scale-up of animal Airlift reactors have been both used and scaled
or plant cell processes, selection of the reactor is up successfully for production of monoclonal
made more complex by the nature of the organ- antibodies (MAb). Birch5 describes the selection
ism. There was an early hope that most, if not at criteria for a system to mass produce MAb:
For personal use only.

all, of the newer, complex biomolecules could be

Unit costs
made in bacterial or yeast cells after appropriate
Economies of scale
genetic transfers. This hope remained a hope and
Aseptic operation
the desire for product led to greater and greater
Ease of process control and automation
use of animal and plant cells, even with the atten-
Mixing of shear-sensitive cells
dant problems. Hu and Peshwa3 present both a
Acceptable mass transfer characteristics
historical review and some projections in the de-
Compatibility with upstream and downstream
sign and operation of animal cell bioreactors. They
processing
discuss the evolution from roller bottles to
Simplicity
microcaniers, then to hollow fiber and ceramic
systems. Scale-up considerations for the various Apparently, vessels of 10,OOO 1 size are in use.
formats are given. Macroporous carriers (examples Problems in handling large volumes of broth are
of structural materials are collagen and gelatin) also discussed. Techniques used include tangen-
are reviewed. Cell culture of some transformed tial flow ultrafiltration,precipitation, ion exchange
cells as aggregates has been successful. A number chromatography, gel filtration, and affinity chro-
of scale-up issues are discussed; the major issues matography. Other workers6 succeeded in volu-
are agitation-aeration effects, oxygen supply, and metric scale-up from 2 to 40 1 in airlift reactors. A
understanding of reaction kinetics. The authors murine/murine MAb was produced in both batch
come to a conclusion that is, in the main, correct, and semicontinuous operation. No problems are
“Despite the great advancement in bioreactor discussed and it seems that the scale-up protocol
developments in the last decade, their operation is went well.
still largely guided by an educated guess.” There Verax’ workers have published extensively
are 122 references cited in the review. on their proprietary mammalial cell-fluidized bed
It should be noted that a not unexpected result culture systems. One article discusses cGMP
of the proliferation of reactor styles is the favor- facilities using collagen microspheres to prod-
itism expressed by commercial interests in pictur- uct various biopharmaceuticals. A key feature

205
 on the reactor is a recycle loop (microspheres An article from MercklO discusses the scale-
remain suspended in the bed and do not enter up of a novel reactor using static mixing ele-
the loop). The loop is used for pH and tempera- ments as cell attachment and cell growth sur-
ture control, DO control, additions, gas ex- faces. The end product was a viral vaccine. The
change, and sampling. Flow is controlled. Scale- design criteria were fairly strict: uniform irri-
up from 1 x 1O9 to 1.5 x 10l2cells per bioreactor gation of cell surfaces, low shear, high surface-
is described. Many production systems have to-volume ratio, surface/material compatibility,
been altered to permit elimination of serum in CIP, SIP, monolayer growth, nonenzymatic
nutrient feed. Product yield for a recombinant harvest, FDA approval, and, finally, scale-up
blood factor rose by 135-fold as scale increased capability. A prototype (5 x 5 em), total vol-
(fluidized bed volume increased 150-fold). For ume of 103 ml, and a production model (20 x
tPA, a bed volume increase of 300-fold re- 20 cm), total volume of 7.26 1 are described.
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sulted in a product yield increase (units are mg/ This is an example of a nonobvious method
day) of 522-fold. Of course, the problems of used to successfully achieve a number of spe-
scale-up are not discussed; however, the suc- cific goals while also allowing extension to
cessful implementation shows what conditions other cell growth systems.
must be satisfied and what parameters require An article by Kearns' compares bioreactor
attention. In another article from the same cor- systems, discusses scale-up problems, and
poration, comparative studies using the same spends some time on integration of the extrac-
hybridoma cell line were performed in differ- tion train. Following the article, there is a four-
ent culture systems.8The systems were suspen- page summary on fermentor and bioreactor
sion batch, continuous (chemostat), and per- design, suppliers, comments, and additional
fused immobilized microsphere. The article characteristics.
For personal use only.

stresses the importance of cell-specific produc- An interesting reportI2concerns a novel agi-

tivity (CSP in units of pg/cell/h). Specific pro- tation system for shear sensitive cells. Two dif-
ductivity of the hybridoma culture (at constant ferent cell systems (one insect strain and one
specific growth rate of 0.04 h-]) was 0.30,0.90, plant cell strain - both susceptible to damage)
1.64 for batch, chemostat, and continuous per- were evaluated in 3- and 11-1 reactors. An en-
fused fluid-bed, respectively. gineering approach was taken and there are
Alternatives do exist, although it is uncer- plots of the impeller Reynold's number vs.
tain as to how much scale-up has actually oc- power number and k,a vs. power per unit vol-
curred. Lazar9 has written a review on immobi- ume. Further, k,a in various cell growth sys-
lization of animal cells in a fixed bed bioreactor. tems was compared with k,a in the novel con-
Animal cells were immobilized in a polymeric figuration; apparently, the new design gives
matrix of polyurethane foam. Lazar lists the two- to threefold increases in k,a. Interestingly,
criteria for supports (true for any sort of immo- surface baffles did not affect power input but
bi lization) : did increase both rate of mixing and 0, trans-
fer. 0, supplementation was used in the
Chemically inert, no reaction with substrate, headspace. Tests in the new system showed
nutrients, products that the cell strains used were less sensitive to
Stable at elevated temperature agitation-aeration than in conventional reactors.
Safe for pharmaceutical products Guidelines for scale-up are also provided. The
Allow cells to grow and otherwise act nor- techniques used can be followed for any agita-
mally tion scheme.
Retain integrity and remain insoluble Animal Cell Bioreactors, a bookJ3published
High surface area to volume ratio in 1991, begins with a historical overview of
Easy and simple handling animal cell bioreactors, has one chapter on scale-
Consistent quality at an acceptable price up (but scale-up is mentioned in many places),
and ends with a chapter on large-scale purifica-
The highest scale described is 2.5 1. tion of clinical products derived from animal cell

206
 cultures. Many experts contributed and although process control, comparing the three classes. The
one must search for problems, many solutions (or article is comprehensive and will prove helpful in
potential solutions) are presented throughout. bioreactor selection. Strategies for the various
Another book on mammalian cell culture, types are described. Seventeen characteristics of
Large-Scale Mammalian Cell Culture Technol- the “ideal reactor” are given. No reactor class is
ogy, is worth reviewing.14 Once again, many ex- “ideal,” but every nonideality presents a potential
perts give both reviews and details of different problem, so the table is a useful starting point.
technologies. There is added consideration (sepa- The authors urge selection of up to six relevant
rate chapters) on process technology management, reference cell lines for use in different systems. In
process validation, and design considerations for this way, research results can be more readily
a manufacturing facility. These latter subjects are compared with one less key variable to consider.
critical to scale-up and those chapters are very An overview of reactor styles, including cell
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informative. retention techniques, is given in Table 6. All cell

Two specialized chapters give details on de- culture systems (all fermentors, in general) have
sign of cell culture reactors and scale-up of cell areas of concern (or in the worst case, problems)
culture systems.15De Bruyne discusses bench scale as listed at the bottom left of the table. Within
reactors, shear, continuous culture, methods of reason, pH and temperature of nutrient feeds to a
oxygenation, and design criteria for a commercial classical fermentation do not present problems.
system between 0.5 and 3 1. Griffiths reviews On the other hand, control within a far narrower
scale-up and density scale-up (in the latter, pro- range is required for nutrient feeds to a cell cul-
cess intensity is increased). A strength of the ture system. Sterility is a concern in any
review is presentation of alternatives. Perfusion bioprocess, but growth rate or type of product
methods are discussed extensively. Various en- formed in a classical fermentation may inhibit or
For personal use only.

trapment methods are covered. One useful table otherwise render contaminant entry a nonproblem.
covers scale-up factors and compares eight differ- The degree of hazard to the same entry is far
ent systems for anchorage-dependent cells and higher in a cell culture system. Substrate-attached
eight systems for suspension cells. The factors are systems have an added set of concerns.
scale-up potential, process simplicity, mass trans-
fer efficiency, aseptic operation, direct monitor-
ing, high cell density, steam sterilizability,down- V. FERMENTATION/CULTURE PROCESS
stream compatibility, substrate reuse, area/volume
ratio, homogeneity/mixing, critical control, and
Problems in scale-up can be subdivided; ex-
continuous process potential. Although qualita-
amples of serious issues in each subset are given.
tive factors are used, such an overview is critical
The fermentation process consists of these key
in process selection.
steps:
Another review article by Bliem et al.16
should be consulted prior to selection of an
animal cell reactor (although the points cov- Nutrient preparation
ered are applicable to bioreactors generally). Inoculum development
Selection criteria are given for various systems: Sterilization
airlift reactors, fluidized and packed bed reac- Selection of parameters
tors, hollow fiber systems, and stirred tanks. Selection of process
Scale-up pros and cons are listed in great detail batch
and there is an analysis of batch vs. continuous semibatch
culturing. There is a comparison (qualitative) continuous
of various factors with three classes of systems: recycle
batch, semicontinuous/fed batch, and continu- refeed
ous/perfusion. As an example, systems falling Monitoring
under “continuous and perfusion” would have the Harvesting
lowest operating expense but the highest cost for Cleaning

207
 TABLE 6
Cell Culture Overview

Culture Mode

Suspension Substrate Attached

Stirred tank Microcarrier (conventional)
Column Microporous materials
Beds
Systems
Roller bottles
Stirred tanks
Airlift (internal draft tube or external circulation)
Bubble column
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Hollow fiber
Ceramic annulus (with or without membranes)
Fixed bed
Rotating discs
Plate type

Cell retention
Membrane
Gravity (settling)
Rotating wire/membrane
Aggregate formation
Internal microfiltration
Dialysis
For personal use only.

Cell entrapment in collagen

Problems

All systems (general) Substrate attached

Homogeneity Perfusion (mass transfer rates)

Impeller shear Shear
Surface finish Equilibrium of constituents
Temperaturea Attachment optimization
PHa Carrier dissolution
Osmolaritya Cell density (homogeneity)
lnoculum state Blockage or channeling
Sterility"
Scaling
Adsorption (unexpected)

a Variable is for both process and nutrient feeds.

Agitation-aeration will be considered sepa- tion. General considerations (any bioprocess)

rately; this combined parameter is not only very should include reputability of supplier, capa-
important, but a great deal of published informa- bility of supplier to deliver, price fluctuations,
tion is available covering many fermentation pro- compositional changes, need for use testing or
cesses. prescreening, potential delivery dislocations,
stability under varying storage conditions, and
A. Nutrient Preparation contamination (both during delivery or during
storage). The latter point refers to asepsis in
There are a number of issues to consider in certain cases but for every raw material, gross
nutrient optimization and raw material selec- contamination is an ever-present hazard. These

208
 potential problems are normally not considered Use testing
in research labs; the problems are occasionally 1. An old example is the screening of
considered in pilot plants. However, one or more cornsteep liquor for use in penicillin
problems invariably occur at production scale. fermentation. Historically, many natu-
One or two cases for each potential should suf- ral products have been screened prior
fice; it is useful to address the situation early in to purchase. This is common now with
the scale-up cycle. animal sera. It may be necessary for
so-called “pure” raw materials.
Supplier reputability 2. Use testing is an added expense and is
1. Resellers may or may not stand be- sometimes short-circuited. Occasion-
hind certain reagents. It is embarrass- ally, there is no problem. More occa-
ing (or worse) to find that technical sionally, there is.
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specifications for a critical ingredient

have changed while the end user has Delivery dislocations
never been informed. 1. Rules exist for shipping certain prod-
2. Companies merge, are bought out, or ucts (as herbicides and pesticides) in
fail. Lack of an acceptable second (or dedicated trucks or, at the least, in
third) source is a problem waiting to trucks or tankcars that are thoroughly
happen. cleaned prior to reuse. This ideal sta-
tus is not always achieved, as failed
Capability to deliver bioprocesses have testified.
1. Some raw materials are sourced 2. Delivery at a preselected temperature
abroad. Regulations change, shippers is often requested. What is often un-
For personal use only.

change, foreign exchange varies, and known is when and how far out of
contracts may be difficult to enforce spec a certain delivery occurred.
in a foreign court. Disruption in air or
sea traffic is common. Extension to the other concerns noted should now
2. Little consideration is given to how be obvious. These general considerations are
much material is warehoused (where? frightening enough, even though all have been
what condition? who checks?) out- overcome (most of the time) in classic fermenta-
side of user control. Real turnaround tion. Cell culture is more sensitive to various
time for a change in demand is essen- upsets and so it is no surprise that problem poten-
tial. tial is magnified for relevant nutrient preparation.
Here one must be even more vigilant. First, highly
Compositional changes purified materials (as hormones, recombinant pro-
1. If a technical data sheet exists, it should teins, growth factors) are often used in biosynthe-
be clear as to who does assays, by sis. Second, endotoxin - both present and in-
what procedure, and when they are duced in process - becomes a new potential
done. Dispute resolution should be problem. Last but not least, both the quantity of
clear (this becomes obvious when items, as well as the quality, is much higher in cell
something costing thousands of dol- and tissue culture. Every screening aspect - as
lars per gram assays “low” in the user’s QC testing, shelf life, use-testing -must receive
laboratory). a heightened awareness. As noted above, the po-
2. Storage conditions vary. Simple items tential problems never vanish; they are merely
such as temperature and humidity must controlled.
be considered. More complex issues
(nearby storage and cross-contamina- B. lnoculum Development
tion) including materials of construc-
tion (tanks, pumps, pipelines) should By now, the importance of inoculum devel-
be considered. opment is well appreciated in bioprocessing. In-
 oculum development receives as much care and same performance as lo%, the lower level
attention as the growth and product cycles; this is is preferred, If frozen cells can be resus-
as it should be. The status or “health” of the pended and used (rather than undergoing
inoculum has a great deal to do with the success a growth cycle), this procedure is pre-
of the production cycle. ferred.
The problems of inoculum scale-up and in- 7. Draw-fill technique
oculum development have been discussed. Some If a semicontinuous process (even for a
of the potential problems will be highlighted once few cycles) is possible, productivity could
again: be enhanced and need for fresh inoculum
reduced. A small fraction of a completed
1. Age of inoculum batch (say 5 to 20% of the total) is left in
During scale-up, evaluate responses the bioreactor; fresh sterile medium is
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when a “young” or “old” inoculum is added to start a new cycle. Such a process
used. does not have universal applicability.
Determine quantitative criteria for opti-
mum transfer time or state.
2. Inoculum hold C. Sterilization/Medium Preparation
If inoculum must be held, what are ap-
propriate conditions? What is maximum This is a massive subject and is covered later
“hold time”? What are criteria for discard in this article. We discuss here the criteria for
of inoculum? These are a few of the ques- media preparation. Every aspect covered has the
tions scale-up runs should answer. potential for problems of severe magnitude. It is
3 . Inoculum stability the height of folly to spend great effort and great
For personal use only.

Plasmid-containingcultures or highly spe- cost on selection and QC testing of the best raw
cialized cells are often unstable. Studies materials and, subsequently, mishandle the ingre-
must be completed to insure minimum dients in media preparation.
variability or no unwanted differentiation, The area selected for media storage, prepara-
but it is also important to find and use tion, sterilization, and transfer to production should
quantitative limits that define “good” or receive the same care and consideration as the
“bad” inoculum. production bioreactor or extraction train. (This is
4. Cross-inoculation highly desirable because the media prep area and
If a fermentation is ready to start and the process receives - at the least -the same regu-
inoculum is lost, it would be useful to latory oversight as the production scheme.) The
know when a production vessel (running media prep area is an integral and critical part of
concurrently) could be used to “cross- the process flow so the conclusion is obvious and
inoculate” the waiting vessel. Added flex- logical.
ibility at this point would be an economic Considerations for problem avoidance are
plus.
5 . Inoculum transfer time 1. Ease of access, ease of cleaning, selection of
Scale-up normally means greater volume wall, flooring, ceiling tile materials
and longer time (compared with lab scale) 2. Level of cleanliness in environment (in some
for process steps. Extremes in inoculum cases, HEPA-filtered rooms are a require-
transfer should be evaluated to set appro- ment)
priate limits. 3. Appropriate lighting
6. Number of transfers 4. Clear and appropriate storage of all needed
Each transfer of inoculum presents po- nutrients, glassware, plasticware, filters, pro-
tential hazards. If possible, inoculum tective equipment
growth cycles and transfers should be 5. Suitable instrumentation (properly certified
reduced. If 2% inoculum results in the and routinely checked) that is cleanable

210
 6. Well-agitated vessels, properly calibrated tion-scale reactors.” Although this statement may
7. In-process quality control (by media prep be a bit harsh, it was made in a 1992 publication.
personnel, when reasonable) in appropriate The author goes on to study gas residence time
area with proper logging and instrument distribution, liquid phase motion, and gives cer-
checks tain circulation time distributions. However, the
8. Refrigerated or frozen storage, when required amount of needed information (i.e., missing data)
9. Appropriate storage (properly labeled) of is stressed.
retain samples. Instructions and control on Scale-up is often related to oxygen supply
discard of retains in place (see section on Agitation and Aeration) and a host
10. Suitable recording devices (if computerized, of problems is related to improper (too low or too
software must be validated) and storage high) dissolved oxygen concentration. It is pos-
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11. Appropriate media transfer methodology sible to review selected publications and come
whether via bottle, large container, or pump. away with an understanding of problems that exist
Proper cleanability and potential scale-up solutions that may be ap-
12. All vessels, bottles, or containers used should plicable.
be of suitable quality, should drain com- A review by Leist et covers the situation
pletely, and should be easily cleanable in animal cell suspension culture. Although the
13. Empty raw material containers (bottles, bags, direct lessons involve animal cells, the indirect
drums, etc.) should be properly stored and lessons have a general applicability. The authors
disposed of or recycled list the chief problems as:
14. Scales should be made of appropriate mate-
rials, should print results (even with com- Implementation strategy of the culture system
puter storage of data), should be suitable for Design of the mixing system
For personal use only.

intended use, should be readily cleanable Asepsis

15. Excellent communication must be provided Physical culture parameters
to all necessary internal contacts (examples Chemical culture parameters
are manufacturing group, QC personnel, Measurement and control
facilities management)
16. All hazardous materials must be properly This fairly well covers the spectrum. The mor-
stored and handled; appropriate personnel phology of animal cells, the complexity of the
protection provided genome, and the greater biochemical complexity
17. Appropriate quality water must be provided. (compared with prokaryotic organisms) are all
A back-up supply should be available for responsible, but the problems look familiar even
emergency use for “older” fermentations. The article does go on
18. Auxiliaries should be appropriate for in- to compare culture methods and touches on scale-
tended use. For cell culture, all autoclaves, up in the various methodologies; there are 191
ovens, glassware washers, steam quality references cited. The authors also detail the many
must be carefully reviewed, selected, and scale-up problems related to serum use. Some are
installed obvious, others less so:

Expense
D. Selection of Parameters/Process Lot-to-lot variability
Product is ill or not defined
The scale-up problems that correspond to Serum may contain growth and metabolism
process or bioreactor selection can be assessed inhibitors
after reading a short quote by Lubbert.” “Unfor- Serum may hinder upstream processing (fil-
tunately, the transport problems, microbial kinet- ters, pumps, sterilization)
ics, and especially their interrelationship are not Serum may hinder downstream processing
sufficiently understood, particularly in produc- Poses infection risk (virus, mycoplasma)

211
 Although serum-free media are preferred, opera- should be evaluated. Both upstream and down-
tion serum-free is often not possible. stream steps should be studied. The review does
Another example of a potential microbial present some of the advantages of insect cells
process is given by Yoshida et al.” A unique compared with mammalian cells. Cell line main-
n-alkane-assimilating bacterium was found tenance (for insect cells) is said to be easier, there
(Mycohacteriurn sp.) that could be grown on is a greater versatility in different suspension cul-
methyl ethyl ketone and use phenol as a raw ture, cells are immortal, there is mild or no con-
material for the conversion. A membrane reactor tact inhibition, gentle shaking (no trypsin) will
was run (with cell recycle) and reverse osmosis detach cells from substrate, and cells are less
plus low-temperature crystallization were used to susceptible to shock. This is quite a list of advan-
purify the product. Hydroquinone was produced tages and certain groups are pursuing ICC in a
in the reactor at ca. 2 g/l. Although actual scale- serious way. It appears that on scale-up, the num-
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up was not tried, a possible scheme (process flow ber of problems or the extent of response by
diagram) is shown with a number of useful and insect cells is less than mammalian cells. Whether
inventive procedures given. the advantages can be taken advantage of on a
There are two other relatively new areas in broad commercial scale remains to be seen.
scale-up where some novelty may be required. It is difficult to limit the number of problems
The problem-definition phase is well advanced on scale-up, especially where animal cell culture
and even some breakthroughs can be claimed. is involved. There are many potential pitfalls and
The areas are plant cell culture (PCC) and insect no small set can be created to cover 90% of all
cell culture (ICC). Many of the scale-up problems potential problems. For PCC or ICC, complexity
are unique to the system but, once again, there is (or lack of knowledge) rises and so does the po-
general applicability elsewhere. Buitelaar and tential for problems.
For personal use only.

TramperZodiscuss PCC in a review emphasizing In two tables, an attempt is made to cover

strategies to improve productivity. The recent areas of concern or, at the least, areas for careful
review states that a 75 m3bioreactor (STR) is in review and selection. The first (Table 7) lists what
use at Diversa (Germany) to produce a PCC prod- might loosely be considered outside or environ-
uct. They discuss alternative systems (as immobi- mental issues. That is, the “inputs” or utilities
lization, gel entrapment, biofilm, absorption, foam needed for successful implementation are detailed.
immobilization, and membranes) with examples A deficiency in one of the specific items listed
from the literature. Not surprisingly, productivity can create a problem. No priority can be estab-
data are sparse. The article does contain a theo- lished without some research data. Some ex-
retical cost comparison for production of amples:
ajmalicine-containing biomass. LipskyZ1presents
a somewhat more theoretical approach for opti- 1. Some cells will not grow on plasticware
mization of PCC processes. He presents various irradiated a second time; others will show
problems and stresses the draw-fill technique for no inhibition after a number of irradiations.
economic optimization. Release agents in the plasticware have no
AgathoP presents both theoretical and prac- effect on some cells and a great effect on
tical considerations in ICC. The listing of consid- others.
erations should be familiar by now. First, role of 2. Changes in glassware washer cleaning agent
nutrients and other media constituents on growth, may or may not cause a later problem.
viability, virus infectivity, and productivity should 3. Change in a sterilizing filter may leave no
be determined. Then, physical requirements (as undesirable residue in a nutrient medium; it
temperature, pH, osmolarity) should be deter- may leave an inhibitor to a different cell
mined. Then, response to transients and stresses strain.
should be known. Stresses include agitation, cir-
culation time, bubbles, indirect oxygen supple- What is clear is that untested changes have the
mentation; finally, different culture conditions potential to cause problems. “Every change must

212
 TABLE 7
Cell and Tissue Culture Problems (Scale-Up)
Raw Materials Glassware Plasticware
quality glass type variability
testing (QC) washing source documents
shelf life drying release agents
CO, distribution endotoxin irradiation
alternate suppliers storage sterility
prescreening reuse
hold conditions coatings

Water Media preparation Incubators

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quality SOPS and records temperature

testing (QC) homogeneity CO, (pressure and Oh)
PM schedule testing (QC) humidity
passivation of piping sterility cleaning agents
records shelf life sterility (HEPA)
storage conditions logs
environment (HEPA) connections
certifications and checks

Facilities and utilities

validation
environmental testing
For personal use only.

cleaning/sanitization
PM schedule
steam quality
data recording

be validated” is not a bad rule of operations or Culture degeneration may be a problem

scale-up. Specific productivity may decline (even with
The next table (Table 8) is modified and ex- no degeneration)
panded after Runstadler. Here the cell culture Asepsis is more difficult to maintain
environment itself is the concern. The various Restart (after unexpected shutdown) is more
“input” factors are detailed. Again, every item complex
listed is not necessarily a cause for serious con- Other feeds must be continuous (or semi-
cern. However, there is a wide span of consider- continuous)
ations that must be addressed, either theoretically Downstream processing must be properly sized
or empirically. Usually less flexible (alternate product in same
Finally, selection of the bioprocess depends equipment)
on the microbial cell, the process itself, existing
equipment, and economics. The batch process has The positives of continuous culture are given
the longest history and is probably the safest, by H o ~ h f e l dHe
. ~ ~stresses cost effectiveness and
meaning that technology is fairly well known and also notes, correctly, that continuous culture is a
is simple. Continuous fermentation has the great- powerful method for process optimization. He
est theoretical advantage but also has detriments notes, “Scaleup of a continuous-culturebioprocess
or, at least, potential downside risks. Some are requires detailed data from process development
and optimization studies which should be gath-
Need for excellent control, probably with re- ered with great care, since a scaleup is only as
dundancy good as the input data provided.” Of course, this

213
 TABLE 8
Cell Culture Environment
(Issues of Concern)

Medium Cell and cell products

Nutrients QC Cell density Cell-cell interactions

Hormones Stability Desired product Preservability
Binding proteins Endotoxin Metabolites Safety
Attachment factors Inducers Inhibitory factors Transformation
Enzymes Minerals Viability (stability) Specific activity
Proteases Extracts Validation

W
Water quality
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by McMaster University on 12/17/14

I I cell culture
optimum
environment

Matrix Culture parameters

Physical In vivo simulation pH Redox

characteristics Homogeneity 0 2 CO,
Attachment factors Scalability Temperature Humidity
Hormones Diffusion Pressure Viscosity
For personal use only.

Binding proteins Polarity Osmolarity Foaming

Enzymes Suspending agents Shear-agitation Homogeneity

Note: Expanded after Runstadler, P. W. et al., Continuous culture with macroporous

matrix, fluidized bed systems, in Large-Scale Marnrnalial Cell Culture Technol-
ogy, A. S. Lubiniecki, Ed., Marcel Dekker, New York, 1990,366. With permission.

is true for batch bioprocesses, too. Hochfeld dis- E. Monitoring

cusses the many scale-up factors that must be
considered. Generation number must be simu- All bioprocesses require stringent monitor-
lated (larger volumes mean more doublings to ing; there are limited process optima and there
reach a desired cell concentration). Maintenance are severe regulatory requirements. A listing of
of steady-state conditions (and problems to avoid) potential culture measurements exists.24Selec-
is reviewed. Potential problems to long-term op- tion is process dependent. A listing of potential
eration are touched on: foaming, gasket and seal problems that require scale-up attention fol-
replacement, wall growth, use of anti-grow-back lows:
tubes, and use of a hooded overflow dam are
noted. 1. Sterility
Intermediate choices involve feeding cycles Instruments, in an ideal world, would be
with or without withdrawal of culture broth. noncontacting and not require broth re-
These are process specific. Invariably, devel- cycle. In the real world, the sensor may
opment time is lengthened due to the complex- have to be sterilized and resterilized. Also,
ity of the equipment and controls. It should be entry should not improve chances for
noted, however, that some of these techniques contamination.
are in use and have significantly enhanced pro- 2. Stability
ductivity. After repeated use (including cleaning and

214
 sterilization many times), does the sensor hold tank to feed the isolation train. Time
maintain response and accuracy? to harvest and hold time are both poten-
3. Redundancy tial problems. Cell lysis is sometimes
Some measurements are so critical (pH in desirable in the hold tank, most often it is
many cases, DO in others) that an in- not. Proper operating parameters must be
cycle failure may result in a batch failure. established.
Should a redundant set of sensors be in- 2. Harvest tank
stalled or can a portable flowcell system The harvest tank may require steriliza-
(attach to sample port) be used as a tem- tion and/or cleaning. Scale-up should es-
porary measure? tablish minimum requirements and pro-
4. CentralLocal control tocols. Special conditions (temperature,
inert gas, additives) may be required in
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If centralized control is used, local back-

ups may be required. Even with PLCs, the harvest tank. Special mixing require-
redundancy may be required so manual ments may exist.
operation might complete a batch even 3. Harvest criteria
after a major failure. Failure mode on Not every fermentation or cell culture
valves is important. Do valves fail open? proceeds in exactly the same way. Mini-
closed? to last control position? some mum criteria should be established for
preset posit ion? harvest; if not met, the batch is discarded.
5. Environment Normally, economics will be a key fac-
Many operations now occur in clean room tor. Presence/absence of contaminants is
spaces. Key variables are particle count, another key factor.
pressure (or pressure differential), tem- 4. Batch discard
For personal use only.

perature, relative humidity. It is impor- In the lab, loss of a batch is disturbing but
tant to establish control and monitoring easily handled. At production scale, there
schemes, a method for data recording and may be severe limitations on a discard.
trending, a recertification program, ac- What acceptable procedures are there for
tion limits, and a program of response. batch discard?

The key question is “What are the critical param-

eters to monitor, to record, to control?” One part G. Other Reviews
of the answer will relate to the process itself; the
other part will relate to the regulatory require- A symposium held about a decade ago cov-
ments for the specific product. Both are equally ers research needs in bioprocessing.2s Surpris-
important. Once the answers are written down, ingly (or perhaps not so surprisingly), many
the program must be accepted internally (mean- needs noted then remain as needs today. There
ing by development team) and should be reviewed are generally analyses of design criteria and
externally (with FDA representatives). Missing a some specifics to consider in downstream pro-
critical control point may not result in product cessing. The needs and problems (which are
failure but it may result in validation failure. true at the research scale, but also true in scale-
up) include:

F. Harvesting Pretreatments
End-product inhibition
Scale-up problems related to harvesting and Product recovery from dilute solution
vessel cleaning may be summarized: Oxygen transfer and heat transfer
Bacteriophage
1. Harvest time including storage (harvest tank) Cell recovery
The bioreactor is not normally used as a Stability of immobilized cells

215
 Problems directly related to scale-up are discussed ments are described. The introduction of a mutant
in the same review (by M. Charles): culture is also included; it is a bit difficult to sepa-
rate the key factors. The article lists a number of
Methods to decrease cell growth (probably references related to classic fermentation scale-up.
meaning reduced maintenance requirement, as A careful study of both scale-up and optimi-
well) while driving energetics to product con- zation for a human hybridoma-producing immu-
centration increase noglobulin M (against P . aeruginosa) was per-
Bulk mixing, including monitoring and control formed by Swiss ~o r k e r s . 2Three
~ bioreactors were
New fermentor concepts used (0.5, 1.5, and 12 1). One of the most impor-
Heat and mass transfer tant considerations in any scale-up is detailed in-
Factors affecting substrate and oxygen transfer vestigation of culture conditions (nutrient and
Investigation of pathways of biosynthesis waste product concentrations and specific rates,
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Bacteriophage-resistant cells pH, dilution rate, temperature, DO level) and re-

Improved product separation and purification sulting impact on cell growth and product forma-
Rheology of broths tion. In these studies, attention was paid to mixing
Reliable, rational scale-up strategies and corre- intensity, DO level, cell content, and viability while
lations optimization proceeded. Productivity and yield
were enhanced; feasibility of large-scale produc-
Problems specific to animal or plant cell culture tion was shown.
are also discussed: Two articles by Ozturk and Palsson2*should
be consulted for examples of necessary studies to
Medium formulation be undertaken prior to or during scale-up. Careful
Factors limiting cell growth measurements were made of 0, uptake, ATP pro-
For personal use only.

Control of product expression duction rates, cell growth, metabolite concentra-

Contamination control and biohazard contain- tions, antibody synthesis, as well as amino acid
ment consumption and production rates. Even if every
Hardware and process design parameters biosynthetic step is not defined, these extensive
Product recovery measurements will serve as a guide for successful
scale-up. In these studies, antibody production
Although nothing noted is surprising, points are was not growth associated for the cell line used;
listed for completeness. only acidic pH and high osmolarity altered the
A certain amount of published information specific antibody production rate. The authors’
exists that gives actual performance in the course aim of improving productivity by understanding
of scale-up. Normally successes are given; proce- the impact of process variables on cell physiology
dures involved give insight into key factors. was attained. A spectrum of process variables
Flickinger et aLZ6describe pilot fermentations at was studied, and both input variables and process
the 120 dm3 scale for toyocamycin (an antibiotic responses are available for effective scale-up and
produced by a Streptomyces culture), The proce- optimization. The work described is an excellent
dures detailed are used to scale the process to example of methods that result in problem
12,000dm3.Sensibly enough, the primary criterion avoidance.
in parameter manipulation was whether antibiotic GriffithsZ9reviews batch and continuous pro-
productivity was increased or sustained longer at cesses for animal cell culture. Some information
a higher level. One end result was that productiv- is given on culture vessels in use for production of
ity could be predicted using oxygen-uptake rate. tPA, IFN, and MAbs. Once again the figure of
Also seed tank inoculum was monitored via CO, 10,OOO 1 is noted as the “largest” unit used for
evolution rate and transfer time was selected based cell-derived pharmaceuticals. Some theoretical
on this parameter. Fluid mixing characteristics in cost analyses are given for batch vs. perfusion
the vessels were estimated in a water system, culture. The numerous advantages in the use of
using a food colorant. Other process improve- porous microcarriers are listed:

216
 Cell densities 2C50 greater than use of solid Though somewhat dated, there is a list of
microcarriers fermentor manufacturers available.32 Vessel
Supports both attached and suspension cells characteristics are given. A list of manufacturers
Suitable for stirred, fluidized, or fixed bed re- (vessels over 100 1) is also given in Appendix C
actors of this article. There is a useful review on overall
Short diffusion paths into a sphere design of a pilot-scale fermentation; many issues
Good scale-up potential are covered which should aid others in design and
Cells protected from shear operation of such a facility.33
Long-term continuous culture possible An earlier book on large-scale mammalian
3D configuration easily derived cell culture covers the state of the art in the early
1 9 8 0 ~Almost
. ~ ~ every one of nine contributions
Two reactor configurations were evaluated relates to scale-up. The strict discipline of the
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by Ryll et al.3nin production of IL-2 using BHK marketplace is indicated by the fact that some of
cells. A small (1-2 1) stirred, bubble-free agita- the firms represented either do not exist or are no
tion system was compared with a dense cell HF longer involved in cell culture. This does not
bioreactor. Media were evaluated that were se- detract from the data, conceptual analyses, and
rum containing and also protein-free. DO was actual operational information (at large scale) pre-
controlled at 40% of saturation. Systems are care- sented.
fully detailed. One- and two-step cell cultivation Although not strictly a scale-up problem, pro-
methods were evaluated. Cost comparison is given cess optimization usually becomes a correlary
for various configurations (medium costs rather problem. In the time interval between discovery
than full operational). Highest cost ($1 1.56/mg) and start of pilot processing, new findings are
occurred with roller bottles and the lowest ($0.751 made that improve the process. Alternatively, in
For personal use only.

mg) with the stirred reactor. Because tissue-like the course of scale-up, slight alterations in inputs
aggregates were noted in the HF system, it is are found to improve the process. The need for
unclear whether sufficient optimization occurred kinetic data can be satisfied while process optimi-
prior to the evaluation. zation occurs. Integration of process changes into
Although review articles do not generally the scale-up program is another issue. One ex-
cover problems in scale-up in a broad way, a ample of fermentation optimization is given by
recent review by Werner et al.31is fairly compre- Yang.ls Another example of optimization (pro-
hensive and discusses economic issues as well. duction of recombinant CD4) can be found in an
Biologic properties of cell culture systems are article by Lazarte et a1.36
discussed with attention to potential pitfalls. Op- After reviewing all available processes and
timum ranges for various parameters are given equipment, one is left with a host of choices, even
as well as flow diagrams for different culture after some selectivity is applied. Are there real-
methods. The key factors in selection of a cell world examples that can be studied, preferably in
culture propagation system are capital invest- a single location? Volume 4 of Animal Cell Bio-
ment, amount of material to be produced, ge- technology3’ has many useful chapters (some of
netic stability of the cell line, reliability of equip- which cover newer designs) but three later chap-
ment and sterility control, and, finally, flexibility ters are reviews for specific bioproducts. One
desired in the production plant. The authors note covers growth and production of HIV, a second
that there are “established technologies” for covers interferons derived from human cells, and
mammalial cell culture up to the 15,000-1 scale. the last reviews manufacture and use of
Flow plans (full plant) are presented for compar- carcinoembryonic antigen (CEA).
ing batch and continuous operations along with Each of the reviews covers a wealth of mate-
potential difficulties with each format. The ar- rial, describes alternatives, and the chapter on
ticle is suggested reading for an overview of interferons includes process flow diagrams and
issues involved in mammalial cell culture and protocols used for various key steps. Problems or
product isolation. issues related to scale-up are summarized here:

217
 HIV Use of continuous cell lines will present useful examples of pay-offs related
after adaptation to attention-to-detail. The article is discussed more
Varying levels of HIV product fully later.
with same HIV isolate
Virus variants present
Changes in cell surface mark- VI. AGfTATlON AND AERATION
ers related to
environmental changes (as Scale-up studies are performed to determine
growth factors and nutrient (with as great a degree of certitude as time and
limitation) money allows) those conditions and parameters
Gas exchange rate that will result in the same process result at a new
Seeding cell density scale (size, mass, or volume). As it happens, one
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Variability in kinetics of virus invariably comes to agitation-aeration as a com-

growth plex, intertwined, and problematic set of param-
Strictly controlled containment eters. The variables are intertwined because ob-
Interferons (IFN) Low titers (leukocyte IFN) taining the same oxygen transfer and dissolved
Optimization of large-scale oxygen distribution requires consideration of both
production (lymphoblastoid aeration and agitation. Even if similar (not iden-
Im) tical) conditions are satisfactory, one must con-
Concentration inducer sider both inputs. Further, if aeration alone is used
Scale-up of anchorage depen- (bubble column), mixing must be achieved even
dent cells (p-IFN) without a mechanical impeller. The power input
Complexity of superinduction to a large vessel (however achieved) impacts
For personal use only.

(P-IFN) mixing (i.e., turnover time), liquid shear rates,

Short-term culture (&IF") and gas mass transfer (i.e., the mass transfer co-
CEA Low product concentration efficient).
(high volumes to process) It is well known that oxygen is sparingly
Microcarrier culture 0, sup- soluble in water and culture media at conditions
ply and harvest method normally encountered in biotechnology applica-
Packed bed nonhomogeneities tions. Growth and/or productivity of microorgan-
Need for low protein nutrient isms is often controlled by oxygen availability.
media Scale-up success depends on satisfying the oxy-
Specificity in isolation gen demand all through the volume of interest. In
general, the dissolved oxygen pattern (and changes
These problems (both general and product- during the cycle) should be replicated at the new,
specific) are covered in some detail. In earlier higher scale. This is not always practical or pos-
sections on reactor design and process selection, sible. This discussion is not meant to imply that
many of the issues mentioned in the three chap- only oxygen depletion is a mass transfer problem
ters cited have been discussed. Others are dis- on scale-up. Certain nutrients (especially those in
cussed later in this article. Real-life examples are trace concentration) can be depleted prior to reach-
valuable for many reasons. Two obvious ones are ing a site within the organism where it is required.
(a) problems or issues of concern force reflection Gas-liquid mass transfer is important in main-
relative to one's own scale-up situation and (b) taining desired dissolved oxygen and dissolved
solutions to many serious problems have been carbon dioxide levels in the culture broth. The
devised. oxygen transfer rate (OTR) and the oxygen up-
Occasionally one can find a carefully written take rate (OUR) may be the same, but this is not
article on design and construction of an actual always the case. The OTR is affected by the mass
facility. A useful article (such as one by Srigley3*) transfer coefficient (k,a), the volume involved
will give many lessons in problem avoidance and (V), and the driving force between 0, concentra-

218
 tion at the gas-liquid interface (c*) and 0,con- Many studies have been performed on mix-
centration in the culture liquid (cL). The well- ing times. Generally, at any reasonable scale,
known formula for OTR is differences (meaning nonhomogeneities) are
noted throughout the entire broth volume. Mea-
OTR = kLax V (c* - c,) surements on mixing time are made by having
numerous sensors (or even a single sensor) in-
The mass transfer coefficient is impacted by stalled and by injection of a measurable entity
power input to the liquid (via impeller and gas (salt, acid, or base) via a pulse or step input.
input), c* is impacted by pressure and pure 0, Change in conductivity, color, or pH with time
supplementation, and c, is impacted by cellular is recorded. An immediate problem, of course, is
demand. It is often hazardous to successful scale- that such measurements are made in an aqueous
up to have c,, at zero or a value below that shown system that may exhibit rheological properties
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by McMaster University on 12/17/14

to be critical in lab experiments. Both k,a and c* different from the fermentation under study.
are impacted by temperature but physiological Perturbing an actual fermentation is costly and
temperature optima are fairly restricted so this the input change may alter the system suffi-
effect is minimal or insignificant. ciently to confuse the study. Past studies have
Many experimental studies have been per- shown, however, that 10- to 50-1 fermentors
formed to relate k,a to other fermentation or cul- exhibit mixing times of 1 to 5 s. Vessels of 1000
ture inputs. These inputs are power per unit vol- to 2000 1 exhibit mixing times of 20 to 30 s. At
ume, superficial gas velocity, volumetric gas flow the scale of 100,000 to 120,000 1, mixing times
rate, number of impellers, and tank diameter. rise to 2 min and beyond. If OUR is sufficiently
Gassed power is different from ungassed power high and nutrient solubility is low (as for oxy-
and correlations for changes are also available. In gen), extended mixing times mean that oxygen
For personal use only.

many cases, predictions incorporate a “constant,” deprivation will occur in certain regions of the
which is often a correction factor to allow data to fermentor broth volume. It should be noted that
fit within a certain size range. A summary is along with potential or real nutrient deprivation,
given by H ~ b b a r d . ~ ~ Othe
v e years,
r it has come to there may be equally serious problems in control
be understood that scale-up of agitation-aeration of pH (nonhomogeneity due to viscosity or ves-
by one technique may not give results comparable sel geometry) and in control of temperature. Note
with another method. That is, all critical param- that heat-transfer area changes with the square
eters are not scaled up in the same way. Broth of vessel dimension, whereas volume changes
density, broth viscosity, surface tension, presence with the cube of vessel dimension.
(or absence) of suspended solids all contribute to The impact of volume (scale) on mixing time
confusing, or at least confounding, the issue. The can be simulated at smaller scales. Careful thought
article noted gives a number of studies and re- must be given to impact of mixing and vessel
views on various scale-up strategies and presents geometry on byproduct removal. In many cases,
the author’s recommendation for scale-up. Gen- CO, is evolved in the course of cell growth or
erally, geometric similarity is followed and (k,a) product formation. In a 10-1 vessel, CO, removal
plant is designed to be equal to (k,,a) lab. Pub- is not normally a problem. In a 50-ft-tall vessel
lished correlations are used to determine air (with overpressure), pressure at the vessel bottom
throughput, impeller RPM, and impeller diam- is different from that at the top. Changes in over-
eter. Power consumption (gassed and ungassed) pressure will impact c* favorably but will also
can be estimated. The manufacturers of mechani- impact CO, solubility. Too high a pC0, or a CO,
cal agitators have a wealth of knowledge on scale- level in effluent gas has had a detrimental effect
up methods and can generate different scenarios on certain biosyntheses. 0, supplementation may
(at different capital and operating costs) based on enhance 0, transfer by raising c*; however, 0,
laboratory or pilot plant data. It is probably worth- supplementation will not enhance CO, removal.
while to contact at least two suppliers for various In cell culture, control of CO,/HCO,- ratios is
estimates and technical advice. critical over a narrow range.

219
 Hubbard et al.40have also considered scale- Some special scale-down techniques can be used
up of a polysaccharide fermentation. Equations to detect production problems or define accept-
are shown that give relationships between gassed able ranges for optimum performance. The tests
and ungassed power, generally for Newtonian include delayed inoculation (add how to maintain
fluids. However, for polysaccharide biosynthe- inoculum), using cross-inoculation, use of
sis, the many effects of dissolved polysaccha- antifoam agents (add rate of addition, time be-
rides on broth properties must be known. Physi- tween addition, total volume, impact on isola-
cal properties include viscosity, elasticity, surface tion), changing back pressure, screening for O2
tension, density, and diffusion coefficicnt; not and CO, tolerance, and testing for sensitivity.
all are of equal impact. Published correlations Consideration of surface aeration at small fer-
for mass transfer in non-Newtonian systems are mentor volume is noted.
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by McMaster University on 12/17/14

reviewed. There appears to be general agree- Singh et aL4*discuss scale-up and optimiza-
ment that keeping k,a constant is a useful scale- tion of 0, transfer in both Newtonian and non-
up strategy (as noted earlier). How to achieve Newtonian systems. Further, the article discusses
this result unequivocally is not readily apparent techniques for continuous optimization of 0, trans-
on scale-up. The authors give a ten-step proce- fer efficiency. An Aspergillus fermentation was
dure for scale-up that encompasses both run in three different fermentors (70, 3700, and
Newtonian and non-Newtonian broths. The pro- 36,000 1). The authors introduce a “mixedness
cedure is used to scale-up a process for produc- index” (I,,,), which is
tion of phosphomannan using Hansenufu holstii;
k,a is kept constant. Im=_
(Number_
of impellers
-
submerged)(
~
Impeller zone volume)~

K. J. Jem4’ considers scale-down techniques (Tank volume)

as methods of generating useful information to
For personal use only.

identify and solve potential production scale prob- and further

lems. Common scale-up criteria are based on tank
geometry, oxygen supply, heat transfer capabil- Im = (Constant)(Tip speed) (Tank Diameter) (T& Diameter)-’’’
ity, mixing effect, and impeller power input. Equa- (Tank Height)
tions are given relating power input, kra, tip speed,
mixing time, Reynolds number, and OUR to pro- No claim to universality of this correlation is
cess parameters. As one might expect, a number made. Still, actual data are used to show how the
of proportionality constants are included; although correlation can be used. The peak OTR was 22
values are not given, some are available in the mmbP and it occurred at the smallest scale tested.
literature, some are available from equipment Correlations are given for Newtonian systems;
manufacturers, and some can be generated if ap- power input due to sparged air is included, Meth-
propriate testing is performed. Aside from agita- ods for agitation-aeration optimization (including
tion-aeration effects, the author notes that certain costs) are given. Different operating strategies are
biological factors that are or seem unimportant at described and a specific optimizing algorithm is
lab scale, can have significant effects on large- developed. The procedures are more complex for
scale operations. The factors listed are culture non-Newtonian systems. In a case given, an opti-
stability, phage resistance, cell and product shear mization routine automatically changes aeration
sensitivity, pellet formation, OUR, heat genera- and agitation conditions to achieve the highest
tion rate, minimum DO, tolerance, and maximum OTR at any point in the fermentation. Presum-
CO, tolerance. As noted, this is not an exhaustive ably, 0, supplementation can be incorporated into
list. Another partial list is given by Jem concern- the model. A cost-benefit analysis would be needed
ing scale-down considerations for optimizing a before such a control scheme were implemented
bioprocess: water quality, sterilization-tempera- on a large scale. It often happens that optimum
ture profile, steam quality, low-cost raw materi- rate or yield is achieved at great expense at pro-
als, serial subcultures, inoculation volumes, and duction scale. Value of the product is a key deter-
pH control agents. These are good places to start. minant as to whether the cost is justified.

220
 Work has been published on animal and plant procedure is usually not costly to evaluate. Also,
cell systems relating aeration to hydrodynamics it is not necessary to operate at elevated pressure
and shear impact. In one such article by Jones et (or to supplement with gaseous 0, for that mat-
al.,43 there are reviews of some of the literature ter) throughout the entire fermentation cycle. It
and an attempt is made to understand the interac- may be possible to program or stage the change
tion of microcaniers and turbulence in an airlift to correspond to intervals of low- or zero-dis-
fermentor and to understand the protective serum solved oxygen concentration.
effect. Other protective measures from the litera- Stirred-tank and airlift tower loop reactors
ture are reviewed. Cumulative probability distri- are compared by S ~ h i i g e r lThree
. ~ ~ different anti-
bution for velocity is shown for a 5% serum so- biotics were synthesized in each system (airlift
lution at a superficial gas velocity of 1.3 cm/s. 0.06 m3 and STR 0.02 m3), and an effort was
Other solutions and other gas velocities are ana- made to optimize medium as well as inoculum
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lyzed. Unhappily, effects of serum concentration quality and quantity. Some adaptation of cell
on hydrodynamics could not be shown conclu- morphology improved performance in the airlift
sively. Although no definitive answers are given, reactor. Results varied with the various filamen-
the methodology is interesting and might be en- tous microorganisms used; insights into potential
hanced or expanded in the future. scale-up studies can be extracted.
The driving force for oxygen mass transfer Okabe et al.46used DO control to success-
can be increased by raising c* (0,concentration fully scale-up an optimized fermentation for a
at the gas-liquid interface). 0, supplementation desired antibiotic. Streptomyces sp. produces
is one method; another is raising the operating cephamycin C as well as the undesirable penicil-
pressure in the reactor headspace. It should be lin N. A complex medium was used and DO was
recognized that other gases (as CO,) will have controlled (varying agitator speed at I v/v/min
For personal use only.

an altered, greater solubility at elevated pres- air). After establishing conditions at the reduced
sure. This latter alternative is detailed in an ar- scale, the process was scaled to 1500 1. Results
ticle by Yang and Wang.@ The article lists a indicated that cephamycin C content was increased
reasonably large number of references related to 1.3-fold and penicillin N was not detected at the
enhanced 0, transfer by conventional and end of the run (this was ca. 7 d, but it was not
nonconventional means and should be consulted present as early as 2 d). One interesting aspect
for that listing of recent publications. In the ref- was use of a transfer technique, that is, after in-
erence cited, both E . coli and Ochromonas oculation of the 1500 1 vessel, broth was trans-
malhamensis (a fragile alga) were studied. For ferred to a 20-1 vessel. Both were run simulta-
both a chemical system and E. coli, normalized neously; in this manner, inoculum and medium
maximum oxygen transfer rate is directly pro- variations are eliminated.
portional to normalized pressure. For these or- Practice and theory are combined by Sumino
ganisms -compared with modest agitation-aera- et al.47Two stirred-tank fermentors (0.2 m' and 2
tion conditions -increasing pressure (and using m3) with two sets of 6-bladed turbine impellers
pressure programming) did overcome oxygen were studied. The well-known sulfite oxidation
limitation. Cell density was enhanced in each method was used and results were compared with
case. Pressure manipulation adds another dimen- published correlations. Interestingly, existing
sion of maneuverability when agitation-aeration correlations were not successful when applied to
constraints already exist. It should be noted that 0,-enriched air. The authors developed a new
many industrial fermentations are operated at or empirical equation that gives correlation coeffi-
near 2 bar. Even if the vessel could tolerate 3 cients of 0.926 and 0.947 for air and 0,-enriched
bar, there may be limitations due to air compres- air, respectively.
sor rating and system pressure drop. Also mov- A scale-up problem in a Micromonospora
ing from 1 bar to 2 bar means p2/p, changes by fermentation is described and solved (Yabannavar
a factor of 2. Moving from 2 to 3 bar changes p2/p1 et al.48).The process was run and considered op-
by a factor of 1.5. Still, as the authors note, the timized at the 10 to 100 1 scale. It was scaled to

221
 120,000 1 scale and there was a noticeable loss of Importance of DO level
antibiotic titer. The first thought was that CO, Mechanisms by which oxygenation causes cell
level was responsible; this was shown not to be damage via sparging in stirred reactors
the case. Small-scale fermentations were run at Whether and how cell damage can be reduced
different pressures. Control was 1.68 atm; other
tests were run at 2.03 and 2.68 atm. Cell growth Headspace oxygenation was used in some cases.
was retarded and titer reduced at 2.03 atm; the Sparger position relative to the impeller was also
situation worsened at 2.68 atm. The change was studied. Pluronic F-68 (at 0.1% w/v) was used in
not due to dissolved CO, or pH, which was kept a limited number of runs. One basic conclusion
essentially constant. The tentative conclusion was (for the TB/C3 cells at the scale used) is that DO
that the high DO level in broth was the cause of levels at 5 to 100% with no sparging have no
poor growth and low titer. Fermentations started effect on culturing. Bubble sparging was always
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at low aeration, reduced agitation, and reduced deleterious at any DO level. As sparge rate was
backpressure (which were changed as the fermen- reduced, growth approached results of the
tation progressed) resulted in reduced lag. The unsparged control. Further, it was found that the
variables were then adjusted to reach DO levels way gas is dispersed has a strong impact on cell
that exceeded the limiting value. Results were growth and viability. Sparge rate alone is not a
equal to control run at reduced scale. The concept good correlator of cell damage. Pluronic F-68
of low initial air or agitation has been used in the addition was protective. Bubble diameter was a
past (especially where redox levels must be kept key indicator (bubbles less than ca. 5 mm are
within an acceptable range) in industrial practice more damaging). Sparging to enhance gas-mass
based on empirical performance. The article cited transfer is feasible if sparge rate is kept low (10.02
presents some explanatory data and the “early vvm) and large bubbles (>5 mm) are formed and
For personal use only.

growth phase” of every culture process deserves not broken up by agitation. Addition of selected
attention. and screened surfactants is also suggested.
Damage to animal cells in bioreactors is re- Authors noted earlier (Yang and Wang) have
viewed by pap out saki^.^^ Theoretical consider- also worked in the area of cell damage due to
ations are explained and available experimental agitation and aeration as well as the interaction of
data are analyzed. Various interactions (bead-to- both. A recent article51gives results of the exten-
bead, bead-to-reactor, agitation-aeration) are dis- sive experimentation. A fragile alga was used as
cussed. The mechanisms are poorly understood, the test organism (Ochromonas malhamensis) and
but responsible factors are considered frequency a number of different cell-counting and viability
of bubble breakup (including coalescence) and measurements were made. The MTT (blue
severity of shear stresses resulting from entrap- formazan color development) assay was used to
ment in draining liquid films or bubble breakup. monitor mitochondria1damage. Vessels used were
Suggestions for problem minimization include 1.1- and 10-1 volume. Analysis of the data led to
design for smaller and more rigid bubbles (i.e., a unified bubble breakup and coalescence model;
avoid coalescence and breakup). “Shear pro- specific cell death rate was linearly related to
tectants” prevent or reduce damage by both re- specific bubble interfacial surface area. There is a
ducing bubble breakup and because cells are not strong coupling or interaction between agitation
sheared in liquid films between bubbles. Simi- and aeration that results in cellular damage. Even
larities and differences between free suspensions though empiricism normally results in designing
and microcarrier culture are reviewed and ana- gas mass transfer systems for cell culture, find-
lyzed. ings in this article can be used to more rationally
Oh et al.50 studied growth of mouse hybri- design scale-up studies.
doma cells at different DO levels and different In spite of the requirement for actual testing
sparging conditions. Bubble/impeller/cell inter- and evaluation of the microbial or cell system to
actions are considered. With the murine cell line be scaled up, many workers continue to develop
studied, these areas were studied: formulations for general use, Both theory and

222
 practice (usually ideal cases) result in correlations properly scaled to vessel diameter. When geo-
of greater or lesser utility. Some recent examples metric similarity was achieved, solvent ratio was
can be consulted for the methodology. Moser et constant and independent of scale when power
a1.52operated a deep jet system using a NaCl per unit volume and superficial gas velocity were
tracer. The “tanks-in-series” model was evalu- held constant. No detail in scale-up can be over-
ated. looked or neglected; unexpected results will sur-
An actual scale-up study involving agitation- prise the unwary.
aeration studies and optimization is reported by
workers at Kyowa Hakko K ~ g y o .The ~ ? organism
is a strain of Rhodopseudomonas spheroides and VII. DOWNSTREAM PROCESSING
the product is coenzyme Q,, (CoQ,,). The spe-
A separate review is probably required for
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cific mutant used provides the best product yield

under a limited 0, supply. Oxygen transfer, CO, problems in scale-up limited to downstream pro-
evolution, and oxidation-reductionpotential (OW) cessing. In a very short general review, E n d e r ~ ~ ~
were key scale-up parameters. Pilot runs (30 1) describes the challenges in scale-up of biopharma-
were performed and specific respiratory rate (0,) ceuticals and discusses “. . .the problem of main-
was plotted against the ORP and intracellular taining (engineering through equipment design)
CoQ,, level as well as cell growth rate. Not sur- the same environmental conditions for all produc-
prisingly, optimum ORP for product formation tion and purification steps in the manufacturing
was not the same as optimum ORP for cell growth scale as exist in the clinical trials scale; and assur-
rate. Finally, data are given for 30 1,40 kl, and 80 ing that those conditions are in fact equivalent
kl fermentors. A close correlation is shown be- (quality assurance).” It is not possible to have
tween respiratory rate and ORP; ORP can be used “the same environmental conditions” at every
For personal use only.

as an effective scale-up parameter. A small side- scale, so it might be better to consider the scale-
light of the article was potential overheating of up challenge as giving assurance that the process
medium at the larger scale (apparently resolved profiles and product profiles and purity are un-
by medium supplementation). changed at production scale. That is, the focus
In recent years, advances in impeller design should be on the product rather than exactly on
have been made. Novel designs result in enhanced environmentalconditions, although the latter “con-
mass transfer at a constant power input when stancy” on scale-up is desirable. In the review
compared with the historically used flat blade, mentioned, only a few unresolved purification
disc turbine. Alternatively, mass transfer could be issues are highlighted:
held at a constant value, whereas a retrofit (mov-
ing to the new impeller design) would result in Cell disruption Longer hold times result
lower energy (mixing) costs. In one such design in increased activity loss
- at a power input of 1.5 HP per 100 gal and a Adsorption processes Extended processing
superficial gas velocity of 6 ft per min -there is times gives undesirable
an enhancement factor of about 15. Actual results side reactions
using one such design in simulated xanthan broth Crystallization Crystal size distribution
are given by Galindo and N i e n ~ w . ~ ~ varies at different scales.
The importance of attention to detail and ex-
actness in scaleup are shown in a paper by Bourne Because there are three dozen (or more) unit op-
et a1.’5 Three vessels (0.045, 0.45, and 4.5 m3) erations or unit processes that can be considered
were run with an 0,-sensitive culture fB. subrilis) part of a downstream bioprocess, there is a great
producing acetoin and 2,3-butanediol.In the small- potential for scale-up problems. The number will
est tank, deviation in solvent ratios was noted; be some multiple of 36. Specific areas of concern
this was related to DO content in the medium. are covered in review articles. The articles should
There was a lack of geometric similarity, that is, be consulted for problems already encountered as
the shaft diameter in the smallest vessel was not well as a number of solutions to some of them. A

223
 list of extraction/purification processes is given in Furthermore, antibody leakage could result in
Table 9. product contamination. Advantages and disad-
vantages of the technology are discussed and
many practical problems (including some rem-
TABLE 9 edies) are reviewed. In spite of the cost of the
Downstream Processing technique, its application may be enhanced by
the nature of biomolecules of interest. There are
Separation Concentration
104 references listed.
Filtration Membrane system Japanese workerP present scale-up data on
(RO/UF) hydrophobic interaction chromatography (HIC)
Membrane system Extraction for purification of a fermentation derived
Floatation Distillation biomolecule. The purification process is rather
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Gravity Precipitation
Centrifugation Crystallization
involved and includes ion exchange chromatog-
Flocculation Evaporation raphy, HIC, hydroxyapatite chromatography, and,
Disintegration Ion exclusion finally, gel filtration chromatography. The ini-
Stripping (vacuum or tial scale used a 16-mm ID HIC column; the
gas) process was successfully scaled to a 1 13-mm ID
column.
Purificatlon
It is also possible to find successful purifica-
Crystallization Lyophilization tion schemes in the patent literature. Merck has
Chromatography Spray drying a European patent59application that is entitled,
Ion exchange Tray drying “Purification of recombinant Epstein-Ban virus
Carbon treatment Vacuum drying antigens from VERO cells, yeast cells or L cells.”
For personal use only.

Electrodialysis
The two major EB membrane antigens, gp 350
Adsorption
Affinity partitioning and gp 200, are synthesized and purified in prepa-
ration of vaccines. The applicants state, “The
Reactionshodifications Final product present invention is amenable to scale-up for
producing large quantities of antigen suitable for
Washing Formulation commercially produced vaccines.” One example
Hydrolysis Dosage form
Immobilization Adjuvants
starts with a recombinant mammalial expression
Racemization Sterilization system, proceeds to cell disruption, one or two
steps of lectin affinity chromatography, gel fil-
tration chromatography, and certain “additional
Yarmush et al.57review immunoaffinity pu- steps,” which may be required. Use of protease
rification. With the advent of highly specific inhibitors is described. Other steps give various
compounds produced at rather low concentra- products that might be used. Although problems
tions, there has been a growth in use of highly in purification are not described, there is a rea-
specific adsorbents. The authors give activation sonable amount to learn from successful tech-
procedures, review coupling chemistries, and niques.
discuss factors that influence stability. Four fac- It is probably a good idea to spend some
tors are listed that can cause loss of column time reviewing general concepts in bioproduct
capacity in the course of repeated operation. separation, that is, reflection and consideration
These are of alternatives should precede creation of a flow
sheet. Too early or too hasty a selection may tie
Nonspecific binding the process to techniques that later prove less
Irreversible denaturation (usually related to than optimum; issues of validation and/or clini-
harsh elution) cal testing may preclude major changes. One
Ligand leakage such overview is given by Fair.60 He uses a
Enzymatic or nonenzymatic proteolysis process engineering approach and begins with a

224
 culture broth containing less than 1 g/l of prod- overall unit product cost, whereas isolation or
uct. Separation system constraints are described, extraction cost made up the other 50%. In the past
but one or more steps consisting of conventional 2 decades, the move toward recombinant organ-
filtration, ultrafiltration, centrifugation, and sedi- isms and far more exotic compounds (produced at
mentation can be used. Product concentration al- extremely low titers) means that the fer-
ternatives are discussed. Product isolation, using mentation:isolation cost split is now more like 1:8
specializedtechniques, are reviewed, as well. After or 1:lO. That is, the culture step might incur 10%
this generalized discussion, more specialized tech- of unit product cost, whereas isolation cost is 90%
niques - with a wealth of published information of the total. It should be clear, therefore, that any
and references - are covered in depth in a slim early choice of an isolation route (especially for a
volume.61There are five long chapters with major regulated product) might well incur heavy later
headings on large-scale gradient elution chroma- production costs. There is a delicate balance that
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tography, membranes (includingreactors and elec- comes into play early in the development cycle.
trically driven membrane processes), aqueous two- On one hand, there is the demand for speed -
phase systems (with lists of applications), affinity this may be related to one or more of need for
interactions, and selected precipitation. Bio- patent protection, the start of animal trials, com-
molecules are discussed throughout, theory is petitive pressure. Although the research scientist
given, optimum strategies are discussed, and many has a very wide spectrum available of devices,
references are included for each subject. columns, adsorbents, and techniques, there is pres-
Although there is not a great deal of pub- sure to “make product.” Yield is not really critical
lished information and data on industrial applica- at this point. The tendency will be to select appa-
tions of newer bioseparations, an article by ratus/procedure that “worked.” Not unexpectedly,
Genentech workers6*is clearly outstanding. There once some product is made, demand escalates.
For personal use only.

is a thoughtful review of various systems with Trying alternate routes (better rate, yield, lower
problem analysis; finally, tangential flow filtra- cost) is discouraged as more testing will be needed.
tion was selected for recovery of kilogram quan- Once again, the tried and true is used. There may
tities of rt-PA. The goals were established at pro- come a time (and this may occur early in the
cessing SO00 1 of conditioned medium per hour development cycle) when validation becomes an
(1.25 x 104 1 to be processed in less than 3 h); issue. At this point in time, changing the purifica-
protein yield was to exceed 99%. Actual equip- tion process means revalidation. This may be a
ment selection is described, design of the full costly and serious hurdle. Therefore, choice of an
scale system is described in some detail, and a isolation route is not a small matter. Alternative
photograph is included. Membrane regeneration procedures (which may or may not include use-
and sanitization is reviewed, again with some testing) should be an early criterion. It is not an
detail. The final system is 180 m2in area; a safety unreasonable suggestion to consider alternate
factor was included. Plots of transmembrane pres- purification paths run in parallel. A small change
sure vs. time are given. One sentence in the article in yield or purity may result; this could have
deserves stress, “The determination of process enormous impact later. Furthermore, selection of
capacities as a function of flux rate under various flexible or multiuse equipment or apparatus of-
fluid dynamic conditions is of vital importance to fers potential for new products discovered in the
the economic implementation of large-scale pro- future. Once validation becomes an issue, having
cesses for industrial use.” Major problems in scale- alternate confirmed procedures or techniques
up result when this point is either misunderstood would be of great utility. Of course, development
or disregarded. costs would be higher. It is likely that as the
It is now fairly well accepted that cost analy- newer bioprocesses mature, final product selling
ses of “older” fermentation processes followed price will become smaller and smaller multiple of
the rule of thumb that divided “fermentation”cost final production cost. An early and thorough analy-
and “isolatiodpurification” cost into a 1:1 ratio. sis of recovery/purification economics is an ex-
That is, the fermentation step cost some SO% of cellent idea.

225
 The general problems that occur on scale-up Mechanically fragile Expensive
are those well known in unit operations; those are Temperature sensitive May be corrosive
changes in flow profile (alterations in ideal plug Quality deteriorates rapidly Environmental
flow pattern), temperature gradients, changes escape
in cycle time (excessive hold times), improper Rheologically troublesome
size distribution on initial column packing,
extent of regeneration, column packing useful These are some of the factors that lead the author
life, change in velocity profile, over or under to state, “Working out a satisfactory separation
heating on drying, and, of course, improper sequence is alone a tedious process; adding the
entry of contaminants, be they microbial or various economic ramifications makes it a formi-
chemical. Time-related variables are especially dable task.” There is also an understanding that
hard to fix on scale-up. If an expensive packing scale-up involves nonideal performance and al-
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is used (with or without special ligands or ways involves a series of compromises. The tech-
adsorbents attached), how is cost per cycle to nical hurdles invariably involve more time and
be determined without knowledge of useful life? money that initially planned.
Even if an ion exchange bed were to be used, Another general review of biotechnology
what provision should be made for complete scale-up is given by Trilli.@Each specific area of
replacement? Number, type, and frequency of concern is covered, even if in a terse way. Sub-
regeneration cycles is of obvious importance. jects include sterilization, agitation-aeration,me-
Running successfully for 4 months or even a dium ingredients, heat transfer, culture stability,
year is no guarantee of process longevity. Only process characterization, and broth rheology. In
actual data (perhaps gathered from a continu- the agitation-aeration section, subtopics include
ously running automated system) will resolve power dissipation, shear rate distribution, and
For personal use only.

the problem. Another problem with some pumping capacity. Nine published correlations
adsorbents is that the adsorbent itself may be between K,a and agitation-aeration parameters
attacked (as by microbial or enzymatic con- are given. An empirical flow diagram is given for
tamination). Preservatives must be added either a generalized scale-up procedure:
during storage or, periodically, in cleaning cycles.
One can imagine the result when the preservative 1. Define set of target values (experimentally
is not removed prior to use or, when washed out, determined)
the preservative is not totally removed. In purifi- 2. Compare with limit values on large scale
cation steps, reagent and water purity are of obvi- (lab and published data)
ous importance; simply specifying the highest 3. Combine these inputs to establish target
grade available may prove both costly and inap- 4. Define operating conditions for small pilot
propriate. The other extreme (“use what is conve- fermentor
niently available”) is not suggested, as perfor- 5. Perform experiments; analyze data
mance may suffer. The appropriate scale-up 6. Define operating conditions for larger pilot
evaluation with selected purity water, reagents, fermentor
and regenerants should follow thorough review 7. Carry out experiments
and some laboratory evaluation. 8. Define operating conditions for production
An interesting overview of biotechnology fermentor
scale-up is given by R o ~ e nAlthough
.~~ more 9. Carry out trials
philosophical in tone than totally technical, a 10. Optimization and process evolution
broad-brush analysis of technology transfer from 11. Production
R&D to large volume production is given. Dif-
ferences and similarities to chemical processing Obviously, if any result is unsatisfactory, re-
are covered. Properties of culture broth are de- turn to an earlier step for reevaluation, a change
tailed (each of which may lead to many diverse in operating conditions, or setting of new tar-
problems): gets.

226
 After a review of bioreactors, bioprocesses, problem and the resolution should be of interest
and downstream processing, it may appear that to the scale-up team. Either the problem may
there is an infinite potential for problems in scale- recur or the resolution is only a temporary fix.
up. As one proceeds in scale-up, it still may ap- The same sort of discussion should occur be-
pear that there is an infinite number of variables tween the scale-up team and the manufacturing
to evaluate and a problem potential exists in each group. As noted, failure analysis is just as impor-
one. In Table 10, some selectivity was used to tant as analysis of successful operation.
limit the number of scale-up problems. These
problems do not cover the potential spectrum but
they have occurred in the author’s experience. As VIII. STERILIZATION AND CLEANLINESS
such, they are probably reasonable places to start.
Should another scale-up program evolve and ex- Mammalial cells or other microorganisms are
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hibit none of these problems, that program is off used for full or partial synthesis of desired prod-
to an excellent start. One other use of the listing ucts. Extreme controls are suggested (and some-
shown might be to review it with research person- times mandated) €or performing the biologic pro-
nel (or persons who have done early discovery or cess and for preparation of the final product.
development). Very often, one or another prob- Throughout the process, design criteria must con-
lem arose and was resolved; both the type of sider the culture (inoculum, cell banks), the pro-

TABLE 10
Scale-Up Problems
Actual Cases
For personal use only.

Conventional systems
Byproduct formation (nonphysiologic forms)
Foaming
Cell lysis (related to hold time)
Odor
Culture degeneration (increase in doublings in inoculum buildup)
Characteristics of process water
Poor temperature control (related to heat load)
Weld problems (contamination)
Bacteriophage attack
Effect of defoamer (conventional and draft tube aeration)
Retrofitting (force fitting of process to existing plant)
Film formation (fouling)
Improper emptying of vessels (NPSH problem)
Aerosol formation

AnimaVplant cell systems

Media stability
Variation in supply of natural product
Improper cleaning agents (volatile and nonvolatile)
Materials of construction (including gaskets and seals)
Use of treatment chemicals (as steam)
Unexpected differentiation
Mycoplasma
Sterility (overall)
Recovery and recycle of suspendinglattachmentagent
Hydrodynamic shear
Product stability (both culture and purification cycles)
Waste streams (hazard)

227
 cess, nearby personnel and the surrounding com- card and repreparation are wasteful. Can the
munity. Gross contamination must be guarded media be resterilized? It is usual to have
against and the contamination potential is bidirec- studies performed on exrreme cases of ster-
tional. Contamination refers to entry of adventi- ilization in the course of scale-up studies.
tious materials, undesirable microorganisms or Resterilization can be performed (on small
their byproducts, foreign chemicals, or leakage scale); process responses can be studied. If
outward of process streams up to and including radiation is used, can culture vessels or media
final product. containers be resterilized, whether single use
Sterilization may be achieved by various containers or recycled containers are used?
means. What is facile at a small scale is often Remember that washing and recycle is dif-
difficult or impossible at a large scale. Examples ferent from single use modes; both should
of sterilizing methods are be validated at extreme conditions (i.e.,
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multiple radiation cycles or multiple auto-

Moist heat Filtration clave cycles). It is hazardous to assume that
Dry heat Chemical agents oversterilization (whatever the technique) is
Radiation Low-temperature plasma “safe.” Sterility can be assured but process
performance cannot.
Problems related to sterilization studies and 3. Changing from one 0.2-pm filter to another
scale-up are described: may be harmless in lab use. (The preferred
course is to validate at every scale.j For cell-
1. Many culture media contain labile sub- culture media at production scale, this may
stances. Autoclaving (e.g., 121“C for 30 min) or may not be harmless. Testing of steriliz-
a 50-ml sample is routine. There is usually ing filters for potential cytotoxicity is cur-
For personal use only.

a rapid heat-up cycle, the 30-min hold fol- sory by the manufacturer. Even if it were
lows, then a relatively rapid cool down fol- not, the cell line and the medium used in a
lows. The time-temperature relation gives new bioprocess were most likely not checked
total heat input. Sterilizing 500 kl of the by the filter manufacturer. Type, size, manu-
same liquid is more complex. Batch steril- facturer should always be validated during
ization involves a long heat up and may scale-up. If the filter is to be reused and
involve steam injection, the 30-min hold is resterilized a number of times, scale-up test-
the same as before, then there is a relatively ing should include a larger number of reuse
slow cool down. The time-temperature rela- cycles than the production process will ever
tion is very different. Total heat input is far undergo.
higher than in the small-volume sample case. 4. Dry heat is used for materials that withstand
Not only can heat-labile substances be lost elevated temperatures for extended periods.
(degraded) but reaction products (as from Elimination of endotoxin is often achieved
the Maillard reaction) might form in appre- concurrently with dry heat sterilization.The
ciable quantities. Continuous sterilization process and equipment should be validated
(so-called high temperature-short time, or for both sterility and absence of endotoxin.
HTST) can shorten heat-up and cool-down In this case, somewhat less severe condi-
times and because hold temperature is nor- tions should be tested (than those to be used
mally well above 12loC, hold time is very in routine processing) and validated. In this
short (it might be measured in seconds or a way, a slight excursion (but within the vali-
few minutes). dated range) would not constitute an “out of
2. In laboratory experiments,an autoclave shut- spec” condition.
down or cycle error normally means prepar- 5. Loading of autoclaves, ovens, radiation
ing a new batch. When large volumes of chambers is a key variable. In sterilizing
valuable substrate are involved and unto- equipment validation, many different con-
ward temperature fluctuation occurs, dis- figurations are tested. Of course, a load

228
 condition more severe than that to be used retrofit facility, quality of steam to be used
is checked. If liquid volumes are to be is a critical point.
sterilized in an autoclave, surface tem- 10. Phage infection may not be a problem in a
perature is not the key criterion. Tempera- lab or pilot plant. Routine processing at pro-
ture (and time at temperature) at the cen- duction scale (large number of batches,
ter of the liquid mass - or at the point in higher volumes, aerosol formation) creates
the mass that reaches minimum tempera- the ideal environment for phage magnifica-
ture last - is critical. Sensors or sterility tion and infection. There are at least two
test items (strip or vial) must be located at counters: design to prevent phage attack and
a number of sites, including the key loca- have phage-resistant cultures available. For
tion noted. cell strains, mycoplasma is an ever-present
6. Sterilization at extreme conditions (what- danger. Extensive screening (at every step
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ever the method) might release agents or of cell bank preparation) and prompt de-
chemicals in the item to be sterilized. The struction of mycoplasma infected banks are
best method in this instance is an actual use countermeasures.
test. That is, sterilize the vessel, container,
tubing, plasticware at an extreme point, then
evaluate utility as well as sterility. Items to A. Aseptic Design
be reused must be evaluated after a number
of cycles greater than that to be used in Aseptic design is a massive subject. Refer-
practice (as noted earlier). ences are available that cover the large number of
7. Membrane filters are in common use in large details to be considered.6s67
volume air filtration. These filters must be Problems in scale-up are both diverse and
For personal use only.

installed properly and must be sterilized complex. Some of the problems can be summa-
under carefully controlled conditions. rized:
Prefiltration is also desirable. In some cases,
condensation is undesirable. Therefore, air 1. All pipes to be used in an installation should
temperature must be well above the dew be sloped to drain. Standing liquid is an
point. invitation to problems.
8. If a chemical agent is to be used for steril- 2. All piping leading into or out of a fermentor
ization, processing should occur at concen- should be steam sealed; for example, they
trations and times greater than, and less than, can be maintained under positive steam pres-
the design criterion, an acceptance range sure when not in use (121°C minimum). It
should be created that ensures: may be necessary to cool the pipe prior to a
Complete sterility over the range transfer in or out.
No degradation or reaction of the desired 3. If possible, use a magnetically coupled agi-
product tator seal (no stuffing box). If an agitator
Complete loss, elimination or degrada- seal is to be used, a double mechanical seal
tion of the sterilizing agent (absence in is suggested. Seal faces should operate at
finished product) high temperatures or have a high-tempera-
No byproducts that are harmful or unex- ture lubricant (examples are steam or sterile
pected defoamer). If a hazardous culture is under
9. If steam contacts a process stream (or sur- study, the mechanical seal should be en-
faces that contact the process/product cased in an outer, emergency seal assembly
stream), the purity of the steam must be (failure of the inner agitator seal will not
considered. There are requirements for pro- cause hazardous spray or discharge).
cess steam in food, dairy, and pharmaceuti- 4. Sensor inserts should be of fail-safe design;
cal facilities. Commercial equipment is avail- an inner seal or O-ring failure is not cata-
able for generation of “clean steam.” In a strophic.

229
 5. If necessary, off gas must be rendered harm- gone a pressurization test (to assure absence of
less by filtration or incineration. pin-holes or breaks) as well as outside steriliza-
6. Valves should not have rising stems; pre- tion. It is useful to perform a pressure test in-
ferred mode is to have no path between the house after a filter has been used. If the used filter
process stream and the environment. fails the postuse test, media filtered should be
7. Inoculation should be affected without ex- refiltered or discarded. As noted elsewhere, any
posure of fittings to the nonsterile environ- change in filter supplier or design or method of
ment. Ideally, any connection made should sterilization should stimulate revalidation.
be sterilized prior to use. C h i s P gives details on design and operation
8. For clean rooms: Proper design and con- of bioreactors with sterility in mind. Actual valve
struction to preset quality level, appropriate sequencing (with appropriateflow diagrams given)
testing and validation, appropriate training, is given to assure complete flushing and heating.
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and monitoring. Inoculum transfer and sampling are covered. The

9. The gas exhaust line of a fermentor must be author recommends against use of flexible trans-
carefully designed. Entry of contaminants is fer piping (need for elimination of air pockets and
possible at this point. Viewports should be condensate pools). In my experience, Teflon@-
provided, sloping is essential, provision for lined, stainless steel braided flexible hose can be
cleaning and/or sterilization of the effluent used routinely if proper fittings and procedures
gas line are all useful considerations. are utilized.
10. Positive pressure should be established and Two other areas will be touched on. The first
maintained in a sterile vessel, a sterile concerns validation of cell banks. When products
transfer line, or a sterile space (as a clean for human use are produced by mammalial cells
room). Development of reduced pressure or when human cells themselves are used (as in
For personal use only.

(as a partial vacuum due to steam conden- tissue engineering products), it is necessary to
sation) could allow entry of undesirable fully validate the cell strains. Whether procedures
organisms. are performed in-house or at outside laboratories,
11. There should never be a permanent connec- work should be conducted under good laboratory
tion between sterile and nonsterile parts of a practice (GLP) regulations of the FDA. Suggested
system. Temporary connections, as for clean- tests include:
ing, are permitted but only when a specific
Tumorigenicity in athymic nude mice
function is being performed; otherwise, dis-
Mammalial cell growth potential in soft agarose
connect.
Evaluation for adventitious viruses (in vivo)
12. All parts of a sterile system should be de-
Evaluation for adventitious viruses (in vim)
signed for ease of cleaning and ease of in-
Evaluation of HIV-1 (antigen capture ELISA
spection. A periodic inspection schedule is
technique)
recommended.
Karyotyping and species identification
13. All instrument holders or vessel entries
Evaluation of cytomegalovirus status (in v i m )
should be sloped to drain. Prevent formation
Retrovirus particle detection by electron micro-
of “dead spaces” or pockets by proper design.
scope
14. Manifolding is permissable but not desir-
Detection of Hepatitis B surface antigen (enzyme
able. Redundant systems or check valves
immunoassay )
should be used to prevent improper transfer
Detection of xenotropic retroviruses (mink S+L-
or cross-contamination.
focus-induction)
Sterile filtration is used more and more as cell Bulk sterility
culture systems are scaled up. There are many Mycoplasma assay
filter manufacturersand many different filter media Detection of Epstein Barr virus DNA sequence in
now available. Some systems permit reuse; others cell genome
do not. Quality control on purchased units is criti- Detection of retroviruses (reverse transcriptase
cal. If possible, select systems that have under- and DNA polymerase activities)

230
 One should review the FDA document Points to time table for all intermediate steps should be
Consider in the Characterization of Cell Lines, established. Any one of the many substeps has the
dated 1987. Preparation of a cell bank for produc- potential not only to create a scale-up problem but
tion use is time-consuming and complex in any to derail the entire development sequence. The
event; the added time and cost in the validation seven general steps are preliminary cell line screen-
effort means that every precaution -taken early- ing, characterization of master cell bank (MCB)
on -will be well worth the effort. The validation and manufacturer’s working cell bank (MWCB),
testing may take 3 to 4 months after test samples in-process testing, validation of the purification
are delivered, and full testing will cost between process, characterization and testing of final bulk
$40,000and $50,000 (1992). Failure in one of the product, studies on toxicology and pharmacoki-
final tests will mean not only waste of a great netics, clinical trials. It should be obvious that the
amount of resources but also delay in scale-up or seven steps could very well be doubled or tripled;
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by McMaster University on 12/17/14

production. the important point is that an overall project sched-

The other issue concerns final product test- ule be established that encompasses all required
ing. The full scope of this subject cannot be cov- testing.
ered here, but it should be obvious that product lot Selection of level of containment required is
analysis is an area that must receive careful and an important criterion from both a cost and an
specialized attention. A short review article69gives operational viewpoint. Table 11 lists various de-
a list of relevant documents issued by the Center sign points or associated points to consider. There
for Biologics Evaluation and Research of the FDA, is a short description for the highest containment
by the European Community and by the Canadian level required, that is, not every rDNA or cell
Ministry of National Health and Welfare. A short- culture production facility requires satisfaction of
flow diagram gives seven general subject head- every design criterion. An analysis of the organ-
For personal use only.

ings; each step is both costly and time consuming. ism and the process will probably lead to un-
Each step must receive detailed attention and a equivocal answers to many, or even most, poten-

TABLE 11
Containment

Highest containment level

Process separated from environment Absolute - use closed system

Exhaust gases Complete treatment - incinerate or filter (full validation)
Seal design Totally enclosed - failure alarm
Valve design Totally enclosed - bellows or alternate
Transfer (in or out) Prevent release - double pipe
Harvest Total cell inactivation by chemical and/or physical means
Spill or leak Containment - emergency response and inactivation
Ambient pressure Lowest pressure (negative to rest of structure)
Safety Posted areas, trained emergency response team
Airlock entry and exit
Special hoods and garments
Access Severely restricted - testing of personnel
Washing/decontamination Essential facilities with required showering
Input and exit air (facility) HEPA filtered
Facility Sealable for total disinfection
Effluent treatment Full containment, complete inactivation
Air testing Routine testing in limited access area, rest of building, and
surrounding environment
Training Complete and documented with periodic
retraining
Samples, sinks, showers, drains Collection and complete inactivation

231
 tial problems. The few uncertainties should be materials compatible with the sterilization
resolved with the aid of experts in the field. It is process?
probably safe to say that the more “cost” is deemed Is there proper inventory and production flow
the key to a judgment call, the more likely regu- control to prevent mixups between sterile and
latory problems (or worse) will ensue. This is not nonsterile products?
a plea for overdesign; it is a suggestion that expe- Is packaging adequate to maintain sterility
rience and expertise (with regulatory input, if re- during acceptable shelf life?
quired) be the keys to an appropriatejudgment on Are chemical and/or biologic sterility indica-
level of containment to be installed. tors in use? Are they appropriate for the pro-
There is a federal guideline on level of con- cess and product?
tainment applicable to good industrial large-scale Is there meaningful testing of product and pack-
practices (GILSP) with emphasis on use of re- aging after sterilization?
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combinant DNA-derived industrial microorgan- Is resterilization permitted? Are adequate

~ (Appendix K, pg. 33175) com-
i s m ~A. ~table records maintained regarding reason for and
pares requirements for 21 design and operating results of resterilization?
criteria. Many design criteria are detailed; the Are cleaners and sanitizing agents approved to
guidelines are not onerous and should be re- assure compatibility and effectiveness? Are
viewed if a new facility is planned or a major residues measured?
retrofit is to be undertaken. Many of the points Is there a proper investigative method to fol-
cover personnel entry and personnel protection; low up on sterility failures?
these points, if followed, could protect the pro-
cess and product as much as they protect indi- These are only a limited number of ex-
viduals. amples. If answers to these and other questions
For personal use only.

were to await planning of a manufacturing fa-

cility, valuable time would be lost. A great deal
B. Regulatory Aspects of experimentation would have to be redone. If
these issues are faced in the laboratory and at
The process and product to be scaled up the initiation of scale-up, valuable information
must pass a host of internal hurdles. Steriliza- can be gathered for both internal use and for
tion - at one point in the process or another regulatory purposes. Further, design of the
(and in many cases, at multiple points) - is manufacturing facility is simplified. It is never
only one of these internal hurdles. If a healthcare too early to establish guidelines for steriliza-
product is involved, government regulation is a tion. Once established and documented, regula-
given. This is true in most countries around the tors will have detailed proof that an effective,
world. Sterilization is one (albeit very impor- controlled, and reproducible sterilization pro-
tant) part of the codes of good manufacturing cess exists. Performance qualification with the
practices (GMP).71 actual process and product involves (1) product
In the US.,the Food and Drug Administra- functionality evaluation, (2) equipment perfor-
tion has umbrella GMPs that cover various medi- mance evaluation, and (3) microbial challenge.
cal products including drugs and devices. Although It is usual to have three identical runs per-
many of the concerns involve the actual manufac- formed. Under present regulations, the ultimate
turing facility, actual equipment, and production responsibility for effectiveness of the steriliza-
personnel, it is possible to select certain questions tion method resides with the manufacturer. This
that are relevant to the laboratory scientist and the does not obviate the need for organized and
scale-up team. Examples are detailed information in these areas:

Is the environment (especially where pilot quan- 1. Type of process used and impact on prod-
tities are made) controlled to prevent undesir- uct, including measurement of resulting
able product contamination from any source? impurities and residues
Are materials of construction and packaging 2. Average bioburden and alarm limits

232
 3. Product load configuration and relevant test- IX. VALIDATION
ing
4. Sterilization cycle parameters (and records) Generic issues of validation are of obvious
5. Sterilization equipment used, including per- importance in any scale-up. Everyone involved
sonnel training should be interested in assuring that the produc-
6. Recertification and preventative maintenance tion process will result in a product/process at-
7. Type, number, location, measurement pa- taining specified levels of yield, rate, and purity.
rameters (destruction rate) of indicators When a potential human or animal drug is in-
used volved, however, regulatory issues are involved.
8. Incubation media and methods for biologic Therefore, validation (involving one or more cen-
indicators used (include negative and posi- ters of the FDA) must have a higher level of
tive controls) priority.
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9. Reference to approved procedures used It is my contention that the later in the devel-
10. Safety level or inactivation level attained opment and scale-up process that validation is-
11. Packaging materials used and labeling sues are considered, the more likely there will be
12. Completely described validation methodol- a time delay in achieving desired goals. Further,
ogy the later in the cycle that a serious problem is
13. Reprocessing found, the more costly will be the resolution.
Although this is not a call for every researcher to
It is not an overstatement to say that if the become a regulatory specialist, it makes good
sterilization procedure is altered during scale-up sense for the members of the scale-up team to
or if information (as noted) is lacking, there is a become conversant with FDA language, termi-
good chance that a separate sterilization scale-up nology, and guidelines. The guidelines - while
For personal use only.

program will be required. The complexity of ster- serving a regulatory function - are sensible for
ilization coupled with its critical importance to those involved in any process scale-up. This con-
product utility indicate that serious consideration clusion will become clear after reviewing many
be given this operation early on in the scale-up of the concepts and suggestions presented by the
process. FDA.
Of course, terminal sterilization is only one One short document (ca. 10 pages) is worth
method of assuring product free of adventitious consulting for a general overview.72The FDA
microorganisms. If the finished product con- definition of process validation is
tains labile materials or living cells (terminal
sterilization not possible), then sterile process- Process validation is establishing documented evi-
dence which provides a high degree of assurance that
ing is a permitted alternative. In some ways,
a specific process will consistently produce a product
sterile processing requires all of those concerns meeting its pre-determined specifications and quality
listed and more; every process step presents a characteristics.
potential contamination hazard. All ingredients
entering the process (media, cells, plasticware, Further,
substrate, packaging) must be shown to be ster-
There shall be written procedures for production and
ile, all surfaces contacting any of these inputs process control designed to assure that the drug prod-
must be shown to be sterile, any intervention ucts have the identity, strength,quality and purity they
(human or robotic) must be shown to cause no purport or are represented to possess.
problems, and multipoint sterility testing for
both environment and process streams must go Although these are eminently sound practices,
on routinely. Bioburden (other than desired they are more than philosophical in nature. Pro-
cells) cannot be low; it must be zero. Many new cess validation is a requirement of cGMP (Cur-
scale-up issues enter the picture. Elimination of rent Good Manufacturing Practices Regulations
steps and process simplification - before and for Finished Pharmaceuticals [2 lCFR, Parts 210
during scale-up - may be critical for commer- and 2111). The guideline stresses the need for
cial success. documented procedures to monitor output and

233
 validate performance of the manufacturing pro- description of the synthesis should be chosen in light
of the controls (specifications and tests).
cesses that might cause variability in characteris-
tics of in-process materials and the product itself.
Documented procedures must be in place to pre- Although these requirements are aimed at
vent microbial contamination and validation of chemical transformation, the projection to bio-
any sterilization process is required. The guide- synthesis is clear and obvious. Fermentors make
lines document speaks directly to the issue of up only one set of equipment that must undergo
scale-up: review and evaluation. Sterilization is only one
operation (albeit a critical one) in the fermentor.
During the research and development (R&D) phase, Product and impurity profiles are important. In
the desired product should be carefully defined in simple terms: there must be fully documented
tenns of its characteristics, such as physical, chemi-
methods and procedures, there must be complete
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cal, electrical, and performance characteristics. It is

important to translate the product characteristics into analytical procedures, complete records, proce-
specifications as a basis for description and control of dures for making changes and assurance (via
the product. Documentation of changes made during written documents) that consistency in producing
development provide traceability which can later be product having predetermined specification is
used to pinpoint solutions to future problems. achieved. There must be a complete paper trail
from start to finish. Not surprisingly,perfection is
An FDA investigator has written that process
rarely achieved.
validation may include development data that
Although the regulatory requirements are
describe the limitations and efficiency of the pro-
sufficient incentives for following appropriate
C ~ S S . ’ ~A narrative covering the manufacturing
guidelines, it should be clear by now that the
process should describe and define the purpose of
correct approach will aid the scale-up process.
each stage and expected quality at each stage.
For personal use only.

Because validation applies to the entire spectrum

Experimental or development batches made by
of systems, process, documents, and programs
R&D personnel determinethe operations described
that support the production process, successful
and supply raw data for process validation. Obvi-
implementation must include all or many of the
ously, then, laboratory and pilot plant notebooks
requirements of the validation process. For a prod-
and records may be reviewed by regulatory per-
uct that will end in human use, the requirements
sonnel. Full-scale production batches should have
are mandated. It is probably never too early to
been run to ensure consistency and to detail why
consider validation activity; at the latest, these
and how the scaled-up process is the same as that
issues should be brought to the fore at the end of
desired and developed.
the preliminary design phase. Of course, valida-
If any further evidence is needed to conclude
tion activity continues through the design phase,
that process validation is (or should be) an inte-
through scale-up, and, finally, through construc-
gral part of the scale-up process, it can be found
tion and start-up. It is obvious that a premature
in another FDA p~blication:’~
selection of culture vessel, separation technique,
In early developmental work, every step would usu- or purification method will mean that revalidation
ally have been examined (at least for extent of reac- might be required if a significant change in the
tion) and every intermediate at least partially charac- process is made. During a well-planned valida-
terized with some estimate of purity. As experience is tion effort, programs are put in place to provide
gained with the synthesis, the critical reaction steps
and intermediates to be monitored are selected. At the
mechanisms for evaluating changes and their
time of NDA submission, in-process control points impacts on a validated process or piece of equip-
should have been selected and appropriate specifica- ment. The evaluation is multidisciplinary; one
tions and tests established to meet the requirements of person or a group alone cannot make the determi-
the regulations. This whole operation is part of the nation.
process validation of the synthesis. The basis for se-
There are many steps in a well-executed vali-
lecting control points and intermediates should be
explained, and the adequacy of the specifications and dation program. Some definitions follow and these
tests to control the synthetic process demonstrated. are both applicable and useful for scale-up plan-
The ranges for the operating parameters in the written ning:
 1. A Master Plan should be prepared. The meets design criteria and requirements for
Master Plan will include the foundation the control of critical operating parameters.
(rationale) for the entire validation program. OQ is used to establish a performance level
It will describe the project, detail training or baseline that provides clear assurance that
schedules, outline basic procedures, set the system can do what it is designed to do.
schedules and resource planning, acceptance When properly documented, OQ can serve
criteria for equipment, table of SOPS, and as a guide to ongoing troubleshooting.
methods involved in actual validation, and, 4. Performance Qualification (PQ) determines
in general, set the tone for the effort. The that equipment consistently meets appropri-
Master Plan defines quality standards for all ate design and QC specifications. Equip-
critical production parameters and details ment, as installed and controlled, must pro-
the efforts needed to support the manufac- vide satisfactory performance throughout the
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turing process. accepted range of operating conditions. Pro-

2. Installation Qualification (IQ) is a process cess Qualification is not exactly synony-
that determines that the system, as installed, mous with Performance Qualification. Nor-
meets requirements of the design drawings mally, three batches of product are run;
and the written specifications. Included are production parameters should be varied (if
checks of purchased equipment and parts, at all possible) to span the proposed operat-
installation checkout documents, and the ing range. PQ is required for critical utilities
actual installation itself. Records (such as (such as clean steam generation, water for
boroscope readings, passivation, radio- injection, certain gases). After Performance
graphic analysis, test coupons) must be made Qualification is complete, Process Qualifi-
at the appropriate time. IQ auditing includes cation is achieved after analysis of the three
For personal use only.

reviews of calibration records, documenta- (or more) batches, including isolation of

tion and maintenance schedules. (See Table product.
12.)
At some early point in the validation process,
but after the Master Plan in complete, it might be
TABLE 12 a good idea to review the plan and schedule with
Installation Qualifications a nearby FDA office. Because there are many
rules and changes are made fairly often, an early
Description of the process and its intended exchange of views will be useful in establishing
purpose
Identification of items requiring calibration
appropriate emphasis. There is no requirement
Identification of items requiring maintenance for such a meeting but early correction is far more
Establishment of the maintenance frequencies efficient than a midcourse correction. The valida-
and procedures tion effort should also encompass review of SOPs
Cleaning procedures (compliance with GMP), emergency shutdowns
Operating procedures; including how to adjust
and reaction routines, preventative maintenance,
the process
Trouble shooting guide equipment calibration and recertification, clean-
Basic procedures for monitoring and control of ing and sanitation, and creation of indexed and
the operation accessible files. If software is involved (monitor-
A spare-parts list to assure that appropriate parts ing, recording, and/or control), it, too, must be
will be available validated.
Documentation that all appropriate manufacturing,
maintenance, and QC personnel have been
A great deal of effort is involved in the course
properly trained to control this operation of construction. The validation group is involved
in vendor audits, compilation of preinstallation
documents,and monitoring of construction.When-
3. Operational Qualification (OQ) determines ever testing is performed, appropriate documen-
that each piece of equipment (individually tation must be prepared. Sensors must be shown
and in concert) operates as specified and to operate within acceptable limits; when neces-

235
 sary, traceability to a NIST standard should be systematic protocol in moving from the lab to
documented. Whenever necessary modification, full-scale production. The rule, “What can go
repair, or recalibration is required, the work wrong, does go wrong” is universally appli-
must be monitored and documented. Finally, cable. No part of the validation effort can be
the prequalification acceptance testing is as- left out without introducing risk. Furthermore,
sumed to be complete and the system or plant when problems do arise or when needed plant
is turned over to the validation team. The vali- modifications are to be made, one should have
dation team can be internal or external (con- a baseline to work from; proper validation pro-
sultants) or a combination of both. The key, vides this background or documentation and
however, is that the validation team must report “recorded experience.’’ Because validation cov-
to a high enough level within the company so ers the spectrum of procedure and equipment,
that needed changes can be made in a timely there are myriad problems that might ensue. In
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manner. The validation effort is time consum- Table 13, there are a number of issues or prob-
ing and costly; recommendations must be acted lems that recur. Although many of these prob-
on to avoid later and larger waste of time and lems have been discussed elsewhere in this re-
money. view, they can be considered validation issues
With the plant complete, IQ, OQ, and PQ in that documentation - addressing these is-
are completed. The calibration program is ex- sues - should be prepared. If approached at
ecuted and instrument calibration is completed the beginning of the validation effort (corre-
before PQ steps. A validation report can be sponding to the end of the preliminary design
prepared at this time. All validation test results phase, at the latest), there is a good chance that
are included, analyses of test data are given, some accommodation or adjustment can be
and the acceptability of the system or plant to made to prevent an issue from becoming a
For personal use only.

consistently perform within predetermined ac- problem.

ceptance criteria is stated. Process Qualifica-
tion follows (or may be included in the final
report). The regulatory guidance concerning X. COMPUTER USE
validation concludes that there are a number of
factors that could render the process “out-of- Scale-up procedures, at best, require rela-
control.” For a regulated product, the process is tively large inputs of manpower and money.
considered “out-of-control” i f Any equipment or technique that would reduce
experimental variability (batch-to-batch varia-
1. The process has never been validated or tion), improve utilization of personnel, create
has been validated improperly. useful documentation, and speed the process
2. One or more parts of the process have would be welcome. Use of computer control in
been altered to an extent that the “new” pilot operations adds value in all these areas. It
process cannot be reasonably expected to would be beneficial if computer control were
perform as it was designed (or as it was available at full production scale, but this addi-
once validated). tion is not critical. For reasons to be detailed,
3. Instrument or sensor recalibration has not computer use at laboratory and pilot scale is
been performed in a competent manner on valuable.
a routine basis. An entire book has been published on com-
4. Operating controls are not being fol- puter control in fermentation processes.75There
lowed. are individual chapters on specially designed
and installed systems at Eli Lilly and Com-
This short review of the validation program pany, Merck and Company, Bristol-Myers,
should show its importance to scale-up. Even Hoffmann-LaRoche, and Smith Kline and
without regulatory involvement, there are ex- French. There is more than enough information
cellent reasons for following a rigorous and to initiate plans for such a system or to modify

236
 TABLE 13 an existing system if cost-benefit analysis points
Validation Issues/Problems in that direction.
The characteristics and values of a properly
Documentation designed computer system are detailed below
Personnel training with some expansion of specific details.
Exclusion of personnel
Complies with filings
Alternate suppliers 1. Monitoring and control (direct ties to sen-
Process or SOP changes sors)
Self-audits Key variables are either monitored, con-
Production monitoring trolled, data recorded, alarm points set
Improper corrections
-either one or all of these functions can
Lack of policy/procedures
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Improper (or no) approvals be programmed and modified.

Simple file retrieval 2. Allowance for improvement/modification
Process variations not included The system should allow for novel or
GLP not followed changing instrumentation, changing pro-
Software cesses, changing raw material inputs, and
Labels
Complaint form and response
a changing regulatory environment. Ad-
Organization chart dition of new instrumentation-to-com-
Incompletely filled forms puter interfaces should be facile and not
Lack of flow diagrams excessively costly.
No revision number or date 3. Networking
Limits of performance not stated
The system should allow relatively simple
Excessive use of “red lined” documents
linking of intraplant and interplant com-
For personal use only.

Facilities and equipment puters. The system should permit use of

Cross-contamination different devices (multiple manufactur-
Microbial contamination ers) to take advantage of specialty units
Endotoxin and various PCs. It would be useful to
Particulates have elements of standardization between
Personnel flow
the pilot system and the full-scale manu-
Environmental control
Erosion and corrosion facturing computer system. Addition of
Key variable(s) control printers, graphics capabilities, plotters,
Solvent reuse tape, or alternate storage devices should
Deficient engineering drawings be simple.
Water 4. Calculation and control of indirectly mea-
Change orders (construction)
Validation program
sured parameters
Quarantine area We can presume that rapid measurement
Product flow of multiple inputs and outputs is possible.
Utilities (additives) Rapid calculation of indirectly measured
Inadequate maintenance quantities should be possible; further,
Cleaning
some change in input (i.e., some element
Recycle (product, air, reagents)
of control) should be achievable. Indirect
Dosage form parameters include respiratory quotient,
Excipient changes heat load (energy balances), biomass,
Stability mass transfer coefficient, and economic
Bioavailability optimization, Data analysis is designed
Impurities into the system; statistical analysis would
Assay specificity
be a useful adjunct.
Deficient test methods
Record retention 5. Complete documentation
Rework A hierarchy of security levels must be

237
 provided. A complete record of changes cation enhanced. Connectivity to manufac-
to the software must be available, as must turing/inventory control software will be
be a complete record of changes to con- enhanced, as well, allowing improvements
trol points and ranges. The software must in scheduling and resource allocation. Eco-
be documented. Process responses must nomic optimization will be part of the scale-
be documented. A complete record of up process with a company-wide perspec-
activities should be provided. Clear and tive rather than a single-process viewpoint.
complete documentation (delineating in- 3. Artificial intelligence (use of expert systems)
teractions between hardware and soft- is common in certain applications; greater
ware) should be provided. use will be made of these capabilities in
6. Automatic and repetitive process control biotechnology processes. “Smart” alarms -
Certain process sequences or process using complex strategies - will further re-
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control strategies should be included; duce the need for human intervention.
others may be added as the need arises. 4. Novel control strategies will be developed.
One example is vessel or equipment ster- Complex interactive systems will be created
ilization, another is inoculum transfer, a as ever more complex sensors are introduced
third might be oxygen supplementation and complex calculations are made that bet-
under defined conditions. Harvest and ter “understand” the process. Solutions of
transfer can be controlled as well as multivariable equations are now possible but
equipment cleaning. will be applied to biologic systems to a
7. Internal system checks greater degree.
The computer system should perform 5. Prediction of (as well as response to) pro-
internal system checks and appropriate cess upsets will be facilitated by computer
For personal use only.

failure modes should be provided. A control. With appropriate inputs, the com-
system failure should not cause a pro- puter system and its affiliated controls can
cess failure; some redundancy (such as be programmed to give an appropriate re-
local control) should be provided. sponse to an anticipated problem. The sys-
8. Transferability (Standardization) tem response to these preventative measures
If a corporate computer is in place, a will be used to moderate or magnify the
link between the pilot plant computer corrective, but before-the-fact, response.
and the corporate computer is desirable.
The transferability to a production com- These advances can be further developed in a
puter control system has already been pilot plant environment and thus can be both the
noted. Standardization allows for fewer result of scale-up studies as well as major stimuli
people required to maintain systems and to more efficient and speedier scale-up proce-
allows technology transfer to occur more dures.
easily and more rapidly (same hardware- There are recent publications that describe
software-operations interface). up-to-date applications of computer logic and
control to fermentation systems. Shi and Shimi~u’~
The current upgrades and future developments utilize “neuro-fuzzy”control in novel experimen-
in computer controlled biotechnology processes tal control systems that relate DO level, substrate
are also important. Scale-up will be facilitated, feed rate, and ethanol production. Pokkinen et
optimization will be speeded, and regulatory con- al.77create a knowledge-based system to act as a
cerns will be alleviated. supervisory system for a computer control sys-
tem. The expert system consists of a shell (user
1. Documented audit trails will be available interface, reasoning control mechanism, mecha-
for both product and process. nism for automatic fault detection, and a tool to
2. Interfacing to plant-scale or corporate level construct a knowledge base) that handles uncer-
computers will be simplified and communi- tainties by fuzzy reasoning both in measurement

238
 and knowledge. The system was tested with real design alternatives will result in a facility that
data; it is said that the expert system could deter- includes few blind spots and fewer undesirable
mine phases of the fermentation and detect faults compromises. At the very least, the jargon that
not deducible from off-line measured data. Early the different groups use will become understand-
correction of faults was possible. The model sys- able at an early stage and individual concerns will
tem was the lactic acid fermentation. The speed be voiced. Although not insuring success in scale-
with which advances in control theory are being up, these exchanges will allow common discourse.
applied to bioprocesses is remarkable. If a novel process is to be scaled-up, test
Brasker et al.78describe a system that has procedures (asepsis, in-process control, QC) may
been designed and installed that includes process be novel. Early design intervention will allow
automation and up-to-date data collection meth- unique requirements or unique facilities to be
ods. Recombinant fermentation processes are “designed in” prior to costly changes. A few ex-
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stressed but design features are universally appli- amples will suffice:
cable.
Yang et al.79present information on com- 1. Which utilities should be manifolded be-
puter-controlled cultivation for the production of cause there are a number of points-of-use?
recombinant human interferon. The authors es- 2. Is emergency power needed? Where and
tablished a nutrient feeding profile that improves how much? The size of an emergency gen-
both plasmid stability (recombinant E. coli used) erator is a key design input.
and overall interferon productivity. All supervi- 3. It is a simple matter to require proportional
sory control tasks and calculations were imple- control, recording plus alarms for every
mented via computer. The optimized process was variable. Can some variables be monitored?
said to increase interferon productivity by 12.9- Can some variables be controlled without
For personal use only.

fold with a 3.7-fold increase in specific produc- recording? Costs are impacted by the choices.
tivity compared with the manual process. 4. What sort of external and internal security
system will suffice? Is round-the-clock cov-
erage required?
XI. CONSTRUCTION CONCERNS 5. What level of cleanliness is required? Again,
it is very simple to call for clean-room con-
It is not a normal procedure to have research ditions everywhere;cost would be quite high.
personnel involved in the planning of a large Degrees of cleanliness must be defined for
biotech manufacturing complex. Normally, re- the various stages of processing. Appendix
search personnel are involved in laboratory plan- B contains one section concerning “Con-
ning and engineers or development personnel are struction.” This is a starter list for construc-
involved in pilot plant design. There is normally tion concerns before and during scale-up;
some overlap between development and manu- the same (and new) concerns arise prior to
facturing groups in the design of a large, produc- and during construction of a full-scale plant.
tion plant. This division of labor is workable but
research personnel might be profitably engaged Construction of any clean-room space entails
in construction details. There are many areas of its own set of specific requirements. Although
design and many opportunities for cost saving there is a federal standard relating to final certifi-
where a research group that has performed the cation, there is no federal standard (as yet) per-
work might have “soft” information to aid in taining to the protocols needed in clean-room
making appropriate design compromises. Alter- construction. As the clean-room space is built and
natively, design difficulties might lead to pointed enclosed, there is, or should be, a construction
experimentation that would clarify or lead to cor- requirement for increasing levels of protection,
rect choices in planning. The interaction between that is, one may begin with booties and bunny
those who have experienced research problems in suits and progress to face masks and, finally, to
synthesis and isolation and those who can offer full operational gowning. As an example, HEPA

239
 filter installation should be accompanied by use pability
of hoods, gloves, bunny suits, face mask, and Allowable fuels, solvents, paints
booties. Final as-built certification requires full Ease of inspection
operational gowning. It goes without saying that Spare pass-throughs (especially in clean
dedicated tools should be used and, at some point, rooms)
tools and instruments - once cleaned - should
remain in the clean room until all tasks are com- Pipindwelding
pleted. There must be written guidelines for each Materials of constructionlsegregation and
construction stage, and the monitor for compli- control
ance cannot be a representative of the subcontrac- Welding specifications
tor doing the actual work. The reason is obvious. Validation (permanent records)
Once a clean room is shown not to satisfy design Supports and insulation
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criteria, it is a bit late to fix blame and find fault Quality control and daily inspection of fin-
with construction protocols or lack of adherence ished welds
to them. Every precaution must be taken before
and during construction to insure “clean” installa- Equipment
tion; correction after the fact is onerous and usu- Connectibility and coordination drawings
ally costly. Accessibility - inspection and maintenance
An article*O covering construction concerns Sealing (no pockets or traps)
relevant to biotech facilities is worth reading, by File history
all parties involved in commissioninga new manu-
facturing facility. The concerns are relevant to a HV ACfductwork
retrofit facility as well. The major concerns are Leak testing
For personal use only.

divided as follows: Filter testing

Sealants
Cleanliness Mapping (location)
Sealing of equipment and piping (precleaned Proper drawings and monitoring
offsite) prior to shipment
Protective package integrity and maintenance Electrical/instrumentation
on-site Lighting
Cleanlinessfor manufacturingand clean-room Facilities management
areas Requirements
Product/process contamination during a ret- Use of universal solenoids
rofit
Movement of workers and equipment There are innumerable issues involved in cre-
ating appropriate equipment, material, and per-
Documentation sonnel flows. Criticality of equipment, inventory
Process validation documentation of parts, redundancy, potential failure modes, and
Validation master plan hazards analysis are but some of the factors that
Installation qualification (IQ) must be addressed by research personnel in con-
Proper certifications and test results cert with designers and manufacturing personnel.
Proper sign-offs Overdesign is not only costly at the start; high
maintenance and replacement costs will continue
Civil/architectural to be incurred. Design effort, construction cost,
Cleanability criteria and instrumentation requirements must be cen-
Wash/cleaning materials and agents allowed tered on areas that have the greatest potential for
Final wall, floor, ceiling finishes gain or loss in routine operation of the biotech
Pass-throughs, piping chases, expansion ca- process; only input from the “process designers”

240
 (research and development personnel) will result studying what has worked and what is permitted
in the optimum facility when built. and follow those practices. The book previously
A good deal of technical detail is given by noted is a treasury of valuable procedures and
S r i g l e ~in~Chapter
~ 21 in a book published in practices.
1990. The discussion covers design and construc-
tion of a mammalian cell-culture plant for pro- XII. ORGANIZATIONAL PROBLEMS
duction of therapeutics. For mammalian cell cul-
ture, the key design factors concern in-process Although this article covers a great number
and final-product sterility (and dosage form), han- of technical considerations, there are organiza-
dling of medium and serum, process equipment tional or psychological issues that should be rec-
and facility design, and potential for biohazards. ognized as well. Technical problems are most
Cell-culture technology is reviewed and differing often soluble; however, a structural problem that
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by McMaster University on 12/17/14

requirements are related to plant-design criteria. is allowed to persist will delay implementation,
The emphasis of the chapter is on the Invitron lengthen the scale-up cycle, and will cause on-
plant and on actual solutions to real or perceived going irritation and economic loss. Further, the
problems. As an example, the number of air subjective issues often weigh more heavily on
changes ranges from 30 per h (less critical rooms) whether or not scale-up is successful in a short
to over 50 per h in rooms used for aseptic opera- time frame. Structural problems that are allowed
tions, Room air pressures, temperature, and hu- to fester will have a negative impact on every
midity of individual rooms are monitored and example of scale-up, even if technical compe-
controlled by a central, microprocessor-based tence is assumed.
control system. Very early- and regular ongoing- Not every reader will be in a position to direct
reviews with the FDA are suggested to agree on resolution of structural problems. There are many
For personal use only.

design criteria. The more novel the process or reasons to understand the situation, however.
product, the earlier the initial review. A point was Knowledge of a problem may allow individual
made concerning picture taking (coded lines and input to alleviate it. Further, there may come a
other critical items covered in the course of con- time when you can have a major impact on reso-
struction) and use of video taping. These tech- lution. The organization/structul problems of
niques were used to give a visual and easy-to- scale-up or technology transfer can be summa-
understand record for later review and certification. rized:
It is never too early to consider personnel
Inadequate documentation by Research
safety and environmental safety. Happily, there is
Inadequate sensitivity analysis
a very useful book that covers many areas of
Inadequate staffing by Manufacturing
concern.81Facility considerations are covered in
Inadequate training or a heightened perception
six separate sections; each is written by knowl-
of sensitivity
edgeable people. The need for early intervention
Misunderstanding of Manufacturing schedules
(before the facility design is fixed) is clear as is
or equipment
the need for expert opinions. Biosafety practices
Lack of a joint (defined) Development team
are covered from different perspectives, and one
Lack of high level sponsorshiplcorporate inter-
section covers a medical surveillance program.
est
Four separate chapters cover considerations that
Lack of defined objective, whether it be com-
would interest and involve regulatory agencies.
mercial or otherwise
(Also see Appendix D.) This entire book is geared
toward problem avoidance. There are many ways Table 14 lists both some of the attributes of
safety and good practice can be violated; resultant structures that function well (“promoters”) and
costs -in harm, money, bad publicity, and legal some of characteristics that cause problems up to
ramifications - are always high. There are far and including failure (“barriers”). Studies have
fewer correct methods and procedures; it is worth been made of what “works.” Successful compa-

241
 TABLE 14
Transfer from R&D to Manufacturing

Promoters
Bilateral champions
Manufacturing involvement (from design onward)
Joint selection of vendors
In-plant demo (bilateral)
Manufacturing dedicated personnel
Joint implementation plan
Past success

Barriers
Inadequate manufacturing staffing
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Inadequate retrofit
Perception of technology
too complex
too fragile
too variable
Disruption of existing schedules
Unclear benefits
Past failures

nies (meaning those that exhibit smooth technol- and established by another group, there is a
ogy transfer and timely scale-up) have experi- natural resistance to accede. If established by
For personal use only.

enced product and process histories that fall in the fiat, resistance is maximized. Early disclosure
“promoters” column. Characteristics are easily of potential problems and of variation in per-
recognizable. Unfortunately, the obverse is also formance can clear the air at the outset; a will-
readily apparent. The barriers to success need ingness to accept joint resolutions and joint
concerted and directed effort; expectation alone inputs (from the onset of joint reviews) will do
will not cause them to disappear. a great deal to improve the perception of the
There is no single key to success, but if one robustness of the technology.
were forced to order the promoters and select the In an introductory section to this review,
most critical, I would opt for “Manufacturing project planning was discussed. Potential pitfalls
involvement from design onward.” If there are were reviewed in outline form. There are ways to
persons from the manufacturing group selected avoid these pitfalls, and methods have evolved to
to assure success (however that is defined) and enhance changes for success. Project manage-
if there is an investment of time and energy ment techniques are well known; these methods
(duly noted in objectives and performance ap- concern all aspects of implementing a novel tech-
praisal), the odds for successful implementation nology. Factors such as organization structure,
become more favorable. Design, planning, imple- technical requirements, cost, and timing are con-
mentation, scheduling, and criteria for success sidered and addressed. Communication channels
must be jointly set and the manufacturing group are established and a formalized system is estab-
must “buy in” to all these concurrent programs. lished to stimulate cooperation and to correct
The need for involvement at the earliest stage is deficiencies in information flow before the project
obvious. is impacted. It is important that the scale-up project
In my opinion, the greatest barrier to suc- be carefully defined and the reason for imple-
cess lies in a misperception of the technology menting the project is detailed. Once these defini-
(in this instance, the process itself). When one tions are in place, organization and planning can
group is given objectives or results achieved be set, detailed planning can begin, and appropri-

242
 ate resources allocated. It is surprising that these Support functions (internal or external to orga-
seemingly obvious steps are not always taken nization)
or that answers to the questions “Why?,” Response to deviations
“What?,” and “When?” are absent or vague. If Definition of success (project termination)
these definitions and planning steps are unclear
or open to varying interpretation, the seeds for The management of process technology is
future discord and delay are sown. After initial critically important. Even with a superbly de-
steps and project initiation, a systematic proce- signed process, it is possible to build a costly,
dure is put in place to check specific and de- defective, and unreliable facility. The many re-
tailed milestones. Both time and money must quired details (and, by inference, the many poten-
be tracked; the key question is, “Do project tial pitfalls) are carefully listed by Bergmann
results conform to goals, plans, and specifica- (Chapter 15 in Reference 14). Because the gen-
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by McMaster University on 12/17/14

tions?” Note that this question can be put to the eral process is cell-culture manufacturing and
design/construction of a physical facility (pilot because the information is so detailed and me-
or full-scale plant) or to achievement of de- ticulous, this chapter is highly recommended read-
fined development results (rate, yield, purity, ing.
throughput, unit cost). Of course, once tracking A general study on technology transfer from
methods are in place, a parallel function is R&D to manufacturing highlights the many pit-
project control. The need for full and open com- falls.82Although any sort of technology transfer is
munication and channeled cooperation is now discussed, anyone who has been involved in bio-
obvious. Unexpected deviations should not re- technology tech transfer will be able to recognize
sult in fire-fighting and creation of ad hoc group- the applicable difficulties.
ings; correction mechanisms should be in place
For personal use only.

prior to need. Personnel responsibilities should

be established for the overall project and also XIII. SUMMARY
established for deviations and untoward re-
sponses. Finally, a definition for the termina- Just as it is difficult to cover all problems in
tion of the project is necessary. A scale-up biotechnology scale-up, it is as difficult to sum-
project has a start, middle, and end. If the end marize the requirements for problem avoidance
is ill-defined, the project will continue with resulting in successful scale-up. First and fore-
mixed interest or support; no one will be able to most, the project team should understand what
assess achievement or make needed adjustments. sequence of steps is involved. In an accompany-
One result will be a waste of manpower and ing table (Table 15), I attempt to show the
money. Another result will be a psychological iterative process that covers successful scale-up
barrier to future multidisciplinary projects. In and successful process development. The starting
summary, project management methods cover point would be a laboratory process or an im-
these various areas: provement concept. The sequence of steps -
with only major headings shown - must be re-
Overall goal as established in multiple detailed peated as often as necessary until the agreed-on
objectives goal is achieved. Rarely is the process “once
Personnel and managerial organization and through”, that is, one proceeds sequentially and
interactions arrives - in linear fashion - at a full-scale op-
Method of implementation (timing included) eration. More often, there is a repeated cycling
Budget (with diversions and alternate paths) until one
Responsibility assignments approaches the goal. Also, the steps overlap and
Communication flow (including meeting min- not every company group is heavily involved at
utes and reviews) every step. The key requirements for successful
Required approvals scale-up (technical competence is assumed) are

243
 TABLE 15
Scale-Up

Research Summary
Technology Transfer Document
Manuals and Procedures
Quality Control

Improvements L
L L
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by McMaster University on 12/17/14

ir

Optimization
Full scale operation Joint planning
Final documentation Scheduling
Audits (by R&D) Capital equipment
Comparison to expectation Build or retrofit
Economics Cultures/cell banks
Acceptance of process Performance criteria

f L.
For personal use only.

Identify and correct deficiencies Initial runs

Newhodified equipment Preparation of batch sheets
Training of operators ttt Validation
Multiple runs Quality assurance
Yield, rate, purity (statistics) Protocol corrections
Long-term contracts (purchasing)

Involved at every step:

Regulatory affairs Internal audits Purchasing
Maintenance/calibration Training MIS

attention to detail, patience, training, mutual un- tally disregarding one or more of the subjects.
derstanding of the shared goal, and some appre- However, insufficient or no attention to one of
ciation of the iterative process involved. these areas merely lengthens the odds for suc-
Even if difficult, one feels obliged to cess in an already complex situation. Should
present a personal list of the requirements and the reader feel that the task is insurmountable,
details for successful scale-up (Table 16). there are some heartening statistics. In 199 I , it
These are obviously mere headings or, per- is estimated that 6.3 billion gal of beer were
haps, titles for specialized reports or summa- produced in the U.S.; the author estimates that
ries. Each of the twenty separate items could 50 to 100 kg of tPA was produced in the same
stand as the subject of an article; some have year in the U.S. The clear conclusion is that
been the subjects of books. It may be that the scale-up of biotechnological processes is
successful scale-up can be achieved while to- both feasible and possible.

244
 TABLE 16
Details for Successful Scale-up

Appropriate management structure

Joint development team
Multidisciplinary inputs
Complete process documentation
Research, process development, manufacturing
Raw materials supply
Complete set of specifications
Defined QC testing and acceptance criteria
Validated cell banks
Multiple storage sites
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Facility design
Meet all internaVexterna1 requirements
Preventative maintenance
Complete records, logs, calibrations
Materials of construction
Impact on process
Ease of cleaning and inspection
lnoculum development
Process optima
Understanding of trends
Sensitivity analysis
Process and environmental monitoring
Sterility control
Appropriate pressurization
For personal use only.

Alarms, responses, records

Agitation-aerationconditions
Stability
Media, cell banks, culture broth, intermediates, cell culture,
product
Scheduling and planning
Loss prevention programs - hazards analysis
Plant support organizations
Responsibility assignments: technical services, engineering, QC
Training and retraining programs
Internal audit program with detailed corrective measures
Redundancy in process design, key equipment, utilities
Effluent or discharge precautions with special treatments
Defined communications path including emergency response

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Bioeng., 40, 806, 1992. 40.
52. Moser, A. et al., Mathematical models for mixing 68. Chisti, Y., Assure bioreactor sterility, Chem. Eng.
in deep-jet bioreactors, Part 1 Analysis, Part 2 Cal- Prog., 88, 80, 1992.
culation of parameters, Bioprocess Eng., 7, 171, 69. Schiff, W., Moore, A., Brown, J., and Wisher, M.,
1991. Lot release-final product safety testing for biologics,
53. Sakato, K., Tanaka, H., Shibata, S., and BioPharm, 5, 36, 1992.
Kuratsu, Y., Agitation-aeration studies on Coen- 70. Federal Register, Department of Health and Human
zyme Qlo production using Rhodopseudomonas Services, NIH, Recombinant DNA Research: Actions
spheroides, Biotechnol. A p p f . Biochem., 16, 19, under the guidelines, Vol. 56, No. 138, July 18, 1991/
1992. Notices, p. 33174.
54. Galindo, E. and Nienow, A., Mixing of highly vis- 71. Nygard, J., Good manufacturing practices - over-
cous simulated xanthan fermentation broths with the view, in Sterilization of Medical Products, Vol. 111,
Lightnin A-315 impeller, Biotechnol. Prog., 8, 233, Harris, L. and Skopek, A., Eds., Johnson and Johnson,
1992. Australia, 1985, 195.

247
 72. Guideline on General Principles of Process Valida- mentation process, Bioprocess Engineer., 7, 33 1,
tion (An FDA Document), Center for Drugs and 1992.
Biologics and Center for Devices and Radiological 78. Brasker, J., Smith, M., and Zavela, D., Batch pro-
Health, Rockville, MD, May, 1987. cess automation applied to recombinant fermentation
73. Avallone, H., GMP inspections of drug substance systems, BioPharm, 4, 28, 1991.
manufacturers, Pharm. Tech., 16, 46, 1992. 79. Yang, X.-M., Xu, L., and Eppstein, L., Production
74. Guidelines for Submitting Supporting Documentation of recombinant human interferon-a, by Escherichia
in Drug Applications for the Manufacture of Drug coli using a computer-controlled cultivation process,
Subslances (An FDA Document), Center for Drug J. Biotechnol., 23, 291, 1992.
Evaluation andResearch,Rockville,MD, Febmary, 1987. 80. Odum, J., Construction concerns for biotech
75. Computer Control of Fermentation Processes, manufacturing facilities, Pharm. Engineer., 12,
Omstead, D., Ed., CRC Press Inc., Boca Raton, FL. 8, 1992.
1990. 81. Biohazards Management Handbook, Liberman, D. and
76. Shi, Z. and Shimizu, K., Neuro-fuzzy control of Gordon, J., Eds., Marcel Dekker, New York, NY,
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bioreactor systems with pattern recognition, J. Fer- 1989.

ment. Bioeng., 74, 39, 1992. 82. Souder, W. and Padmanabhan, V., Transferring
77. Pokkinen, M., et al., A knowledge based system new technologies from R&D to manufacturing, Res.
for diagnosing microbial activities during a fer- Technol. Mgrnt., SepVOct 1989, p. 38.
For personal use only.

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 APPENDIX A

TYPICAL REVIEW AREAS (SAFETY AUDIT)

1. Hazard classification and HAZOP study

2. Volume of hazardous material (flow plan)
3. Containment practices - all streams
4. Analysis of failures (controls, earthquake, utilities)
5. Training (new employees, ongoing recertification, emergency responses)
6. Facility procedures and practices/Response teams
7. Raw material storage, sample storage, hold tanks, spill control plan
8.
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QA/QC/Sterility control
9. All effluents and aerosols (location, quantity, tests)
10. Waste treatment, including potential for breakthrough
11. Disposal (plasticware, gloves, equipment, etc.)
12. Certification and validation (especially frequency)
13. Documentation (process and procedures) and JSA
14. Emergency procedures/Monitors/Drills/FirstAid
15. Environmental testing and monitoring/Alarms
16. Vaccination program/Serum sampling
17. Medical record keeping and surveillance
18. Self-audit with corrective measures (feedback)
19. Accident and near-miss reports (with follow-up)
For personal use only.

20. Conformation to regulatory standards (EPA, OSHA, TSCA, FWRA, USDA, FDA)

APPENDIX B

DETAILED QUESTION LIST

(Scaleup Check1ist)

Process
1. Is there a reproducible process?
2. Are impacts of upsets known?
3. What is the projected production rate?
4. Are critical control points known?
5. Has a cost of goods analysis been made?
6. Do SOPS exist for the process?
7. Have Research, Development, QA/QC, Engineering, Manufacturing personnel signed off on
process documents?
8. Is all equipment in place to produce material? Are spares or standby units in place?
9. Are cGMP procedures in place (if required)?
10. What is the percent running (or “on”) time?
11. What is expected reject rate?
12. Has a hazards analysis been performed?

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 13. Are cleaning procedures known and documented?
14. Is inoculum development optimized, understood, reproducible?
15. Are all effluentdwastes (gas, solid, liquid) detailed and provisions for handling clearly under-
stood?
16. Is an effective process control and monitoring system described?
17. Are operations sequenced and scheduled appropriately?

Raw materials
1. Are all raw materials and suppliers known?
2. Are alternative sources known?
3. Do QC specs exist on all raw materials?
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4. Are purity requirements known?

5. Are purity and sterility requirements known for biological items?
6. Do contracts exist (if needed)?
7. Are supplies of raw material sufficient (inventory, market conditions)?
8. Any special precautions or special tests needed?
9. Are any use tests required?
10. Do adequate areas exist for storage of raw materials?
11. Can inventory of raw materials be monitored and controlled easily?
12. Are expiration dates known (shelf life)? Can materials be rotated properly?
13. Does each material have its appropriate sterilization procedure?
For personal use only.

Quality controllquality assurance

1. Are all quality control tests (including in-process) known and documented?
2. Are all QA procedures known and documented?
3. Is equipment in place and validated?
4. Are cGLP procedures in place (if required)?
5. Is documentation sufficient to satisfy regulatory needs?
6. Are there special (controlled) areas for “Hold,” “To be Tested,’’ “Approved for Use,” “Approved
for Shipment,” “Reject” for raw materials, WIP, finished goods?
7. Are time sequences known for analytical effort to “clear” raw materials, WIP, product?
8. Are trained personnel available to perform assays?
9. Is there a documented complaint-handling procedure?
10. If outside testing is needed, is time provided? Are costs known?
1 1. Are documentation and record-keeping procedures in place?

Documentationlvalidation
1. Is process documentation complete? System for changes including approvals?
2. How are documents maintained? How are batch records checked and maintained?
3. Who performs certifications? Schedule of preventative maintenance and recertifications?
4. Are internal audits in place? Scheduled?
5 . Is all paperwork (or data storage) easily accessible for regulatory review? Complete trail of process
and product?
6. Are all appropriate permits and licenses in place and available for review?

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 Construction
1. Are designers and contractors aware of facility requirements? Are they prepared to modify usual
techniques and protocols to fit aseptic and/or cleanroom design?
2. Are persons designated to check deliveries of pipe, tubing, ductwork, valves to be sure that (a) end
caps or other protective closures are used, (b) items are stored in a protected location, (c) items
are moved without losing integrity of protection?
3. Are strict rules in place (and enforced) for limiting areas for smoking, eating, drinking?
4. What design details have been incorporated to limit dust and debris and, more important, to limit
introduction of foreign items into hidden areas of the new construction?
5 . Is the construction sequence in place to achieve final design criteria? Do pipes slope correctly? Is
cleanability considered at each step? Are protuberances limited or eliminated? Are openings
sealed or caulked (sanitary and cleanable sealer)?
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6. Are appropriate surface finishes (floor, ceiling, walls, insulation, ducts, passthroughs) selected with
care for the intended use? intended cleaning? antimicrobial properties?
7. Does design account for heat and humidity generated?
8. Are welding specifications sufficiently detailed? Are weld tests (including visual records) in place?
Are welds checked daily?
9. Are appropriate supports in place? Are they cleanable? Are they accessible? Are utility inputs
accessible?
10. Is emergency power available? Are all critical utilities or components correctly connected?
11. Are all HVAC filters prevalidated? Are all HEPA filters prevalidated? Is location of every filter
(every one numbered) known? Are all tests documented?
12. Are all fixtures, fittings, conduits designed to minimize or eliminate contamination and permit
For personal use only.

sanitization?
13. Are documentation procedures in place? Are appropriate certifications required? Are tests defined
(and who performs tests)? Are certified as-built drawings required?
14. Are all pressure vessels properly coded? Are appropriate relief devices in place? Design drawings
properly stamped?
15. Is there provision for complete sanitization (or sterilization, where appropriate), for access for
replacement or for maintenance, for inspection by regulatory persons?
16. Have the designer, contractors, or subcontractors designedbuilt biotech facilities? Can these
facilities be inspected?
17. Are special company personnel (meaning company paying for facility) selected and trained to
inspect work as it is proceeding?
18. Are local, state, and federal regulations known and understood? Are all regulatory issues under-
stood at the outset? (Examples are emissions, fume hood operation and use of radioactive
compounds, hazardous waste storage and disposal, alcohol storage, narcotic storage, handling of
blood or tissue, flammable reagents, special alarms, OSHA rules, boiler operation, recombinant
organisms, transformed or transfected cells, TOSCA rules).
19. Are pipes, tubing, valves, breakers, instrument lines properly labeled, tagged, or identified in a
uniform and consistent manner?
20. If double containment is required, is the design both functional and economic? Are appropriate
sensors in place?
21. What control points or environmental conditions are alarmed? What is alarm function, messaging,
and proposed response?

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 APPENDIX C

MANUFACTURERS OF FERMENTORS
(OVER 100 LITERS)

APV Crepaco LSL Biolafitte, Inc.

8303 West Higgins Road 719 Alexander Road
Chicago, IL 60631 Princeton, NJ 08540

Applikon Lnc. Lee Industries Inc.

1165 Chess Drive P.O. Box 688
Foster City, CA 94404 Phillipsburg, PA 16866
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Artisan Industries New Brunswick Scientific

73-T Pond Street 44 Talmadge Road
Waltham, MA 02254 Edison, NJ 088 18

B. Braun Biotech Paul Mueller Co.

999 Postal Road P.O. Box 828
Allentown, PA 18103 Springfield, MO 65801

Chemap, Inc.* Pope Scientific

111 D Corporate Boulevard N90 W14337 Commerce Drive
For personal use only.

South Plainfield, NJ 07080 Menomonee Falls, WI 5305 1

DCI Inc. Porton Instruments/LH Fermentation

600 North 54 Avenue 3942 Trust Way
St. Cloud, MN 56303 Hayward, CA 94545

Endotronics (AcusystTM) Precision Stainless

8500 Evergreen Boulevard P.O. Box 668
Minneapolis, MN 55433 Springfield, MO 65801

Engineered Technologies Corp. Sulzer Biotech*

222 Metro Center Boulevard 230 Crossways Park Drive
Warwick, RI 02886 Woodbury, NY 11797

Feldmeier Equipment Inc. Walker Stainless Equipment Co.

6800 Town Line Road 618 State Street
Syracuse, NY 13211 New Lisbon, WI 53950

Henderson Industries
45-T Fairfield Place
West Caldwell, NJ 07006
* MBR BioReactor AG (Sulzer) and Chemap AG (Tetra Laval) merged as of November 1, 1992.

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 APPENDIX D

STATUTES AND NOTICES THAT MAY BE APPLICABLE TO SCALEUP

OF BIOTECHNOLOGY PRODUCTS

Federal Food, Drug and Cosmetic Act (FDCA)

21 U.S.C. $8301-92

Federal Register, Department of Health and Human Services, NIH,

Vol. 51, No. 88, May 7, 1986 Notices with Additional Actions of
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Aug. 24, 1987 July 18, 1991

July 29, 1988 Oct. 15, 1991
Oct. 26, 1988 Nov. 21, 1991
Mar. 13, 1989 Jan. 28, 1992
Mar. 1, 1990 Apr. 22, 1992
Sept. 12, 1990 Aug. 26, 1992

Public Health Services Act (PHS), 42 U.S.C. 5526263.

Toxic Substances Control Act (TSCA), 15 U.S.C. 552601-29.

For personal use only.

Occupational Safety and Health Act (OSHA), 29 U.S.C. 55651-78.

Federal Insecticide, Fungicide and Rodenticide Act (FIFRA)

7 U.S.C. $5136-136y.

Clean Water Act (CWA), 33 U.S.C. 55466-466g.

Federal Clean Air Act (CAA), 42 U.S.C. 597401-7642.

Resource Conservation and Recovery Act (RCRA)

42 U.S.C. $56901-87.

Virus, Serum and Toxin Act (VSTA), 21 U.S.C. 55151-58.

National Environmental Policy Act (NEPA), 42 U.S.C. 554321-70.

Comprehensive Environmental Response, Compensation and

Liability Act (CERCLA), 42 U.S.C. 559601-57.

Other acts relate to meat and poultry inspection, plant pests, weeds and plant quarantine. This listing
refers only to Federal jurisdiction and is NOT meant to be complete. Involved Federal agencies
include FDA, FSIS, APHIS, EPA, NIH, and OSTP.

253