aragen.
com
Case Study
An optimal upstream
process development
approach to increase the
titers of the monoclonal
antibodies (mAbs)
A comprehensive upstream development strategy must be devised at both the bench and pilot scales to
develop the therapy in sufficient quantity and purity for regulatory approval and, eventually,
commercialization. The decisions taken during upstream development, such as clone selection, DOE
optimization, media and feed selection, and bioreactor scale, will ultimately determine the titers and purity of
the biologics. This case study demonstrates how Aragen leveraged its almost two-decade of biologics
knowledge to assist the client in developing an effective upstream process development strategy for a new
mAb, resulting in a more than 100 percent increase in titers.
The project
The client was screening a specific Monoclonal Antibody (mAb) against a species of bacteria that causes several
difficult-to-treat infections such as pneumonia and cystic fibrosis lung infection. This mAb candidate was being
developed as a first-in-class adjunctive therapy. Aragen was approached to identify the high producing clone and to
develop an upstream process development protocol for achieving greater therapeutic titers at different bioreactor
levels, streamlining GMP manufacturing.
About the client
The client is a late-stage clinical development company leading the creation of transformative, first-in-class
anti-infectives for life-threatening viral and bacterial respiratory infections. The company’s pipeline of novel mechanism
antibacterial and antivirals, sprung from its proprietary technology platforms, are designed to combat the growing
public health threat of viral pandemics and antimicrobial resistant (AMR) bacteria.
Why Aragen?
• Over 20 years of expertise in cell line engineering and upstream process development that yields higher
results and supports regulatory filings.
• Experience of working on a variety of biologics and building their effective delivery roadmaps.
• World class infrastructure and high throughput analytical facilities at Morgan Hill, California spread across
47000 sq ft that allow us to upstream a variety of biologics in a timely manner.
• A robust IT infrastructure ensured complete data protection and IP security.
Aragen’s approach
A multipronged approach was adopted to identify the high producing clone among the three developed by the client
and to establish the optimum experimental parameters for upstream process development both at shaker flask and
bioreactor levels. The cell line development team worked closely with the client’s technical team for the tech transfer.
Aragen’s bioprocess technologists validated the optimum parameters at bench and pilot scale and all the analytics was
performed by the developability team ensuring an unfailing and scalable upstream process. The process was
developed in accordance with appropriate standards and was in accordance with industry best practices. All the
complications and the associated risks in the developed process were consistently sought out and rectified to ensure
the quality certified process to the client.
Abstract
The process development began with tech-transfer of three RCB
clones from the client's laboratory to Aragen's. To identify the
superior clone first step was to increase the quantity of all the wt
h 5.4
three clones i.e., to generate the development cell banks (DCBs). Gro
6%
11
Titer (g/L)
Initial shaker flask studies were conducted with all three DCBs
using media, feed, and experimental conditions like those of the
client and based on metrics such as protein titer and other 2.5
physicochemical characteristics one clone was selected for
master cell bank (MCB) development. Media and feed screening
were performed on the selected clone using a shaker flask, and Original Titer Titer after PD
eight different media were tested.
After extensive DOE in shaker flask, screen assessment, and parameter optimization, the process was translated to pilot
scale bioreactors (2L). Paraments were further optimized in pilot scale and the product was analyzed for its
physicochemical characters using high throughput analytical techniques. Optimised parameters were tested for
scalability in 7L bioreactor and finally the optimised upstream process was transferred to client's preferred CDMO. The
total increase in the titer after the bioreactor evaluation was equivalent to 116% with titer improving at every stage of
the process development.
Upstream process development for Novel Cell Lines
Data collection from Design of experiments
the sponsor and Process Parameter Technology transfer to the
Technology to identify the optimal
and Screen Evaluation client’s preferred CDMO for
Transfer reproducing the experimental parameters GMP manufacturing
experimental findings and analytics development
Optimizing the growth Pilot scale verification in a
Media and of the clones in different Bioreactor 2L bioreactor. Scalability
Feed Screening feed conditions and Evaluation testing of the optimized
choosing the right clone parameters in 7L bioreactor
Process Development
Step 1: Technology transfer
• Tech-transfer of three clones (C1, C2, C3) from the
client's laboratory to Aragen's facility at Morgan Hill, Fig. 1 - RCBs Viability Data
California and expanding them into DCBs.
90 C1
C2
• Seeding the cells at 0.5 cells/mL every third day for 12 C3
days and evaluating the cell density, proliferation, and 60
viability data.
30
• Result: All three clones showed a cell viability of 90%
to 95% (Fig.1). Titers for all three clones (Fig.2) were
comparable to those at client’s lab. Highest titer of C2 4 8 12
~2.5g/mL. Doubling time (Fig. 3) of all the clones was Time (Days)
equivalent in both labs.
• Duration: 5-6 weeks
Fig.2 Titers Comparison Fig. 3 - RCBs dT
Control Control
2.5
Time (Days)
Aragen Aragen
g/mL
C1 C2 C3 C1 C2 C3
Step 2: Feed screening and selection of a clone
• Evaluation of different feeds for effects on productivity of the different clones.
• Titer of all three clones nearly doubled with cell boost feed.
• However, Efficient Feed C (EFC) feed had a titer that was almost equal to the control feed.
• Based on affinity data, potency testing and Aragen’s experimental finding, clone C2 was selected for
developing the upstreaming process.
• Duration: 4-5 weeks
Step 3 - Media and feed screening for the selected
clone
• Performed design of experiments and shaker flask
studies to identify suitable media, control feed, th 4.4
Grow
Titer (g/L)
feeding period, temperature shift, and shaking 76%
speed.
2.5
• Estimation of titers was done using two different
methods i.e., HPLC and ForteBio.
Original Titer Titer after Screening
• Duration: 3-4 weeks
Design of
Experiments
Feed Media Screening Temperature Feeding Shaking
Selection Shift Strategy Speed
Media, Feed and Process Process development in Shaker flask Protein Analytics
Parameter Screening and expanding high producing clone DLS, HPLC, SEC, ForteBio, AUC
Step 4 – Process parameter and screen evaluation in 2L and 5L bioreactor
• Using ActiPRO and BalanCD medium, eight investigations carried out in 2L bioreactors using cell boost
feed
• Observations: Acti-prio medium produced greater
titers, sodium butyrate did not appear to promote
expression, and lower RPMs were linked to higher
titers wth 4.7
ro
%G
Titer (g/L)
• Further evaluation in 7L bioreactor to ensure further 88
scalability of the process.
2.5
• Duration:
• process parameter screening in 2L bioreactor: 3
weeks Original Titer Titer in 2L BR
• optimization in 7L bioreactor: 2 weeks
Parameter Optimization RPM
in shaker Flask
Temp
Shift
Parameter Optimization
in 7L BR
Parameter Optimization Dissolved
in 2L BR Oxygen
Technology Transfer to
Harvest GMP Manufacturing
Day
Project outcome
15%
The total increase in the titer of the harvest was equivalent 7%
10%
% Increase in the titer
to 116% with titer improving at every step of the process
development.
The efficient process development developed by Aragen 60%
was submitted to the client for scaling up to GMP
manufacturing. Subsequently, the client successfully
conducted Phase I trials. Original Titer Feed Media and Parameter Parameter
Screening feed Screening Screening in BR Optimization
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