Cell Line
Development Services

Stable Cell Line Development at Aragen – Accelerating speed to IND

Efficient cell line development is critical in bringing biologics to market and requires a team of experienced scientists, a portfolio of well validated cell line platforms, and state of the art facilities. Aragen delivers this combination and has completed more than 200 cell line development projects, with over 100 of those cell lines in the clinic following an investigational new drug (IND) application. More than four of Aragen’s cell lines are producing marketed products.

Support for a broad range of host cell lines and expression vectors

Aragen’s researchers are specialists in handling a wide range of host cell lines (CHO, SP2/0, and NS0) and expression vectors (DHFR, Glutamine Synthetase (GS), and antibiotics).

Optimized cell line development services for therapeutic biomolecules

• Global cell line development: USD 11.6 billion by 2028 (Global Market Insights Inc.)
• Due to patent expiry of branded therapeutics- Increased demand for Novel Anticancer drugs, monoclonal antibodies, Biotherapeutics, Vaccine production
• Partnering to generate biosimilars with demonstrable structural equivalence to that of innovator drugs (like post-translational modifications-glycosylation)
• Possess validated CHO-DG44 and CHO-GS cell line and vector combinations with an innovative clone selection process.
• Achieved increased titres up to 6g/L; 33% reduction in CLD timelines; Transfection to RCB in 18 weeks; Royalty-free platform.
• Optimized the manufacturing scale-up with tight control and tailored analytics to ensure that the biosimilar fits innovator drug specifications.
• Working with small to large clients developing biosimilar products for inflammatory disease, genetic disorders, and cancer.

CLD Process Flow

RapTr2022 with Higher Titre and Shorter Timeline – A royalty-free CHO-DG44 CLD

Key Highlights

Our Biologics team has produced a range of biologics, including human, mouse, canine and feline IgGs, fusion proteins, enzymes, hormones, cytokines, mini bodies, and bispecific antibodies with this platform. Major advantage of RapTr2022 is best suited to produce difficult-to-express proteins. 

A strong track record in successfully engineering cell lines to develop the proprietary CHO-DG44 platform for timely delivery of more than 100 CLD projects. Majority of these CLD projects resulted in IND applications leading to clinical development. 

Our resources and capabilities will fit your unique requirements at each step of the CLD process. Our RapTr2022 platform should expedite your biotherapeutic product journey to clinic since this platform minimizes risk and maximizes efficiency.

CHO DG44

Our internal CHO DG44 platform is an essentially free-to-own (royalty-free) and is a high- productivity CHO cell line option that can deliver >4g/L in 5 months for a range of biologics. The DG44 platform has an extensive regulatory track record and uses commercially available media and feeds.

Highlights

• Royalty-free CHO DG44 platform 
• Production of 4-5 g/L protein in ~5 months
• Multiple expression systems (GS, antibiotics, etc.)
• Extensive regulatory and clonality expertise
• IND- / BLA-ready packages
• 50% antibodies
• 50% non-antibody proteins
• Host cell lines include CHO-DG44, CHO-GS, SP2/0
• Expertise in generating Biosimilar Drugs

CHO GS Platform with Higher titre and shorter Timeline for Antibody Production 

Now, increasingly, the GS-CHO expression system, which is protein-free-adapted CHO-K1-derived cell line that employs the glutamine synthetase (GS) gene expression system is used for biotherapeutic production. 

• A royalty-free CHO GS Cellline Development (CLD) platform
• CHO GS- stable cell line expression platform to produce proteins and antibodies. 
• Stable bulk pool (BP) evaluation reached ~700mg/L.
• Mini pool (MP) generated, and MP reached up to 4.2g/L.
• SCC were generated from top 2 mini-pools and fed-batch shake flask reached up to 6 g/L.
• Single cell clones showing stability over 60 days in respect to cell growth, maintaining high viability and specific productivity.
• Using Aragen’s optimized process from transfection to Single cell cone evaluation completed in 18wks.

Sigma’s CHOZN

Sigma’s CHOZN platform is based on deletion of the CHO glutamine synthetase (GS) gene with Talon gene editing technology. The resulting GS-/- CHO host and expression vector with GS selection is a great combination for companies with an interest in an established GS selection system. Regulators are familiar with its efficacy and operation, and its expression vector produces titers equivalent to the DG44 platform.

Mitigating risk with parallel cell line development

Deciding what CLD platform would work best is challenging. Therefore, Aragen’s cell line development service provides cost-effective approaches to test different platforms simultaneously without delaying IND filing timeline.

A poor expressing product is a suitable candidate for testing on multiple platforms. Simultaneous testing provides a mitigation strategy to the risk of having to repeat CLD due to low titers or poor product quality that results in high material costs (COGS). If two platforms have comparable titers, then the superior product quality or reduced milestone costs can drive the platform decision. We have optimized breaks in the CLD process so that parallel work can be stopped as soon as data is available to select the most effective platform.

Case Study : Biosimilar Cell Line Development: Trastuzumab

• Generated clonal cell lines
• Evaluated in Fed-batch Shake flask
• Reproducible data in Bioreactor
• Analytics to meet the analytics

Process Development

A commercial-scale biologic production is only possible when bench-scale prototypes are scalable to pilot-scale and high-volume bioreactors. Parameters should be established both at the shaker flask scale and at the larger scale reactors so that wastage of valuable resources and time are saved. Therefore, it is critical to establish the production parameters following rigorous experimental design and verification of as many variables as feasible. Process development must also include parameter testing at various bioreactor volumes, which is expensive and can cause considerable delays.

• Use of nonproprietary commercially available media and feed for screening studies allows transfer to any CDMO
• Media and feed screening in Shake Flasks
• Process parameter screening and optimization in 1L Bioreactor
• Process consistency and scalability happens in 5L Bioreactor

Process Development (PD) Case Studies: PD to Increase titer & Improve Product Quality

Case Study : Low expressing Recombinant Protein with cleavage issue

• Improved titer from 20 to 300ug/mL with >85% fully formed protein
• Established robust USP and DSP for client

Case Study : RCB titer >5g/L

• Established robust USP with final titer >5g/L where client came with <2g/L

Case Study : SP2/0 Cell line with inconsistent, unscalable USP

• Improved titer with consistent scalable USP and transferred to CDMO

Case Study : Biosimilar SP2/0 Upstream Process Development

Upstream Process Development

• Media adaption
• Shake flask fed batch evaluation with different media and Feed
• Scalable and consistent data from shake flask to 1L and 5L Bioreactor in terms of VCD and titer profile
• Glycan profile, SEC, cIEF, Mass Spec, SPR is comparable to Originator (golimumab) 

Part A: Media Adaptation; Growth Profile

• Master Cell Bank (MCB) received from Client was thawed, scaled up in the original medium, Media A, based on Aragen protocol
• SP2/0 cells were adapted in different medium. The SP2/0 adapted well (viability >90%) with Media B, D and F. These medias were used in the next stage

Replacement Name
Media A-control
Media B
Media C
Media D
Media E
Media F

Replacement Name
Feed A-control
Feed B
Feed C

Part B: Media & Feed Screening

PVCD: peak viable cell density

Part C: Shake Flask Evaluation Optimization

• With the temperature shift to 32°C, cell viability was enhanced, prolonged evaluation with cultures lasted longer than 10 days
• Media F: Peak VCD was observed at 14-17x106cells/mL with the viability better maintained and productivity was highest

Part D: Bioreactor Parameter Screening 

1L BR Cell Growth Profile – VCD and Viability

• Cultures lasted from D10 to D14
• PVCD ranged from 14 to 20X10⁶ c/mL
• Higher rpm and 40% Do worked better to achieve higher PVCD. Till D6 viability were maintained higher
• Temp shift to 34⁰C showing sharper drop than TS 32⁰C
• At 40% viability cut off criteria 50% couldn’t reach D12 and 50% showing better viability on D12
• Seems 250rpm worked best

1L BR Metabolic Profile 

Glutamine was maintained till D4 and that helped the cells to reach desirable PVCD. Other than scheduled feeds few additional Glucose feeds were added. Overall glucose level maintained within normal range. For all 6 BR conditions lactate level was 4g/L. BR3 & BR4 showing ~2g/L. With 250rpm all BR showing less than 6mM NH4+ accumulation.

1L BR – pH, PCO₂ and Osmolality 

• pH set point with wider dead band helped the cell growth with minimal base addition
• PCO₂ was maintained ~20-30% for all cases
• Osmolarity for all cases were in acceptable range

1L BR Harvest Titer

• Harvest titer ranged from 343 to 884mg/L.
• Highest titer by BR4.

1L BR Summary Table

1L BR Analytics

• Requested Analytics:
  • ProA Purification & A280 Titer
  • SDS-PAGE
  • SEC-HPLC
  • cIEF
  • Glycan Profiling
  • Intact Mass by LC-MS

1L BR Analytics – SDS-PAGE Non-Reduced

1L BR Analytics – SDS-PAGE Reduced

1L BR Analytics – SEC, cIEF, LC-MS, and Glycan Profile 

• BR4 showed similar profile in SEC-HPLC, cIEF, and LC-MS to the originator. 
• Glycan profile needed to be lowered, thus in optimization glycan were modified using galactose and uridine

1L BR Summary

• Culture duration lasted from D10 to D14
• PVCD reached 13 to 20×10⁶ cells/mL.
• Metabolic profile is within the usual range
• Harvest titer reached similar or a little better than shake flask evaluation
• Successful scaleup from SF to BR
• From screening parameter, the best condition is from BR4-
  • DO- 40%
  • Agitation- 250rpm
  • TS-32⁰C
  • pH set point 7.2+/- 0.4

Multiple Il Bioreactors were run to optimize the reproducibility of the process.

Upstream

Upstream Summary

• The process is reproducible (BR1 is like the control).
• Earlier TS on D3 with same feeding did not reach the expected PVCD (BR2), however with increase feed on D5 & D7 could reach the expected PVCD(BR3).
• Higher feed on D5 and D7 with the same TS on D4 couldn’t keep the cell’s viability high. There was a sharp drop in viability after they reach the peak (BR4).
• Addition of the Glycan modifier didn’t show an adverse effect on cell growth (BR5 & BR6). However combined feed showed sharp drop in viability after D7 feed (BR6).
• For the next part or BR Optimization, repeated the run for BR5 (Galactose) and BR6 (Galactose & Uridine). 

Downstream and Analytics

• BR1 and BR5 is from the initial 1L BR Optimization

Downstream and Analytics Summary 

• From BR process optimization experiment BR5-2(1LBR @0.1% Gal Plus) showed reproducible data as the control with modification of Glycan profile. It needed a little more optimization of the glycan profile.
• For the next 2X5L BR run was designed to optimize further glycan profile to match the innovators lot as well as reproduce the same data as control run.
• BR1: Gal Plus @ 0.15%
• BR2: Gal Plus @ 0.2%
• Earlier BR5-2(@0.1% Gal Plus) was used as control for this set of data.
• Galactose + Uridine did not improve the glycan profile or the titer, thus Uridine is removed for the next stage.

Bioreactor Repeatability & Scalability

5L BR Growth Profile – VCD & Viability

• Both BRs showing similar growth profile like control.
• Cultures lasted from D14.
• PVCD reached ~16X10⁶ c/mL for both BR.
• Both BRs showing similar viability profile as control.
• Harvest viability on D14 is ~20%.

5L BR Growth Profile – Metabolic Profile

• Glutamine was maintained till D4 and that helped the cells to reach desirable PVCD
• Glutamate level is very similar for both BR runs 
• Cultures never ran out of glucose
• Other than scheduled feeds no additional glucose feeds were required for both BRs
• Lactate level was 2-3 g/L for both
• Both BRs showing similar NH4 accumulation
• Final NH4 was less than 7 mM

5L BR – pH, Gas, & Osmolality

• Osmolality was comparable
• PCO₂ was maintained ~20-30% for all cases
• Higher pH set point with tight dead band and later pH shift helped the cell growth with minimal base addition

5L BR – Summary Table

• Both BR showing similar growth and viability profile as control
• Data is reproducible and process is scalable (1L BR to 5L BR)
• Using galactose with 0.15% and 0.2% did not impair the cell growth, viability as well as titer
• Harvest Titer is comparable for both 5L BR to the Control

5L BR – Analytics Summary

5L BR Glycan Profile

Glycan profile for 5L BR1 and BR2 improved the glycan profile to be comparable to the innovators.

Case study – Adalimumab (Humira) using Aragen’s proprietary GS Vector

• Stable bulk pool (BP) evaluation reached ~700mg/L
• Single cell clone (SCC) from BP reached up to 2g/L with clones showing stability up to 60 days
• Mini pool (MP) generated, and MP reached up to 4.2g/L
• SCC were generated from the top 2 mini-pools and fed-batch shake flask reached up to 6 g/L
• Single cell clones show stability over 60 days in respect to cell growth, maintaining high viability and specific productivity
• Using Aragen’s optimized process from transfection to Single cell cone evaluation completed in 18wks

Stability study of Single Cell Clones (SCC) from Bulk Pool (BP)

• Completed stability study on top 3 SCC from BP
• Completed 60 days stability both with and without selection.
• High viability, good growth rate, shorter dT
• All three SCC showed stable specific productivity up to 60 days

Typical Instruments utilized for: Media and feed screening, process optimization in bioreactor and scalability

To Increase the Titer & Robust scalable USP Development

AMBR250

Key Highlights

• Ambr250 is a bioreactor system for parallel fermentation or cell culture development with intuitive programming and maximum flexibility.
• Equipped with fully automated liquid handling platform.
• The Design of Experiments (DoE) software allows you to complete the process design for up to 24 fermentations with wide range of cells, at a time with enabled investigative capability.
• The High Throughput provides an efficient and rapid scale up system to explore a wide range of conditions and strains with scalability to both bench top and larger stirred tank bioreactors.
• It provides high precision environment to support the demands of mammalian cultures.

Productivity

Drastically improves productivity and enables full DoE experiments to be performed at a fraction of the cost compared with traditional bench top reactors.

Versatility

Provides a highly parallel system to rapidly develop processes for clones or strains in wide range of cells in bioreactors that have full individual control of culture conditions.

Scalability 

Optimum scalability can be obtained due to presence of bioreactor vessels that are geometrically like larger bioreactors. Furthermore, all processes on the system that correlate empirically to that of the large bioreactors.

Convenience 

Fully integrated to sensors that eliminates long hours the users spend in the lab, manipulating, cleaning,  or setting up the experiment. For more information, contact bd@aragen.com