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.
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).
CLD Process Flow
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.
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.
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.
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.
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: Epidermal Growth Factor Receptor Blocker (HER-2 Type) or Rec-MC Antibody
|IgG Epidermal Growth Factor Receptor Blocker (HER-2 Type) or Rec-MC Antibody|
|Sample ID||Main Peak||HMWS||LMWS|
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.
Case Study : Low expressing Recombinant Protein with cleavage issue
Case Study : RCB titer >5g/L
Case Study : SP2/0 Cell line with inconsistent, unscalable USP
Case Study : Biosimilar SP2/0 Upstream Process Development
Upstream Process Development
Part A: Media Adaptation; Growth Profile
Part B: Media & Feed Screening
|Media A||Media B||Media D||Media F|
|Culture lasted||7-9 days||8-9 days||7-8 days||7-8 days|
|PVCD range||5.5-6×106 c/ml||4-4.5×106 c/ml||6.6-9.6×106 c/ml||11-13×106 c/ml|
|Titer range||330-500 mg/L||180-280 mg/L||400-500 mg/L||400-500 mg/L|
PVCD: peak viable cell density
|Tested Media and Feed||Outcome||Scenario|
|Media B||Culture lasted similar to control/orginal (Media A), PVCD and tier lower than Media A||Remove|
|Media D||Culture lasted similar to control, PVCD and titer better than Media A||Potential media|
|Media F||Culture lasted similar to Media A, with highest PVCD and titer is also a little higher||Potential media|
|Feed B||This feed showing better than control feed in all cases||Potential feed|
|Feed C||Similar performance to control feed||Remove|
Part C: Shake Flask Evaluation Optimization
|ID||Run Duration Days||%Viability at Harvest||PVCD E6 c/ML||Harvest Titer (mg/L)|
Part D: Bioreactor Parameter Screening
1L BR Cell Growth Profile – VCD and Viability
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
1L BR Harvest Titer
1L BR Summary Table
|Summary Table for BR Screening Parameter|
|BR ID||PVCD||Run Duration||Harvest Viability||Titer by BLI mg/L|
1L BR Analytics
1L BR Analytics – SDS-PAGE Non-Reduced
Band at the expected size for all BRs, same as the Originator
1L BR Analytics – SDS-PAGE Reduced
Two bands at the expected size for all BRs, like the Originator
1L BR Analytics – SEC, cIEF, LC-MS, and Glycan Profile
|Sample ID||ProA||SET-HPLC||cIEF||LC-MS||Glycan Profile|
|% Recovery by ProA||Main Peak%||HMW%||LMW%||pl||%Main Peak||%Basic Peak||%Acidic Peak||ME Da||G0F||G1F(1,6)||G2F||G2FBS1|
1L BR Summary
Multiple Il Bioreactors were run to optimize the reproducibility of the process.
|ID||PVCD X106 c/mL||Run Duration Days||Harvest Viability %||Titer by BLI mg/L|
Downstream and Analytics
Summary of Bioreactor sample Optimization
|Protein A Drip Column Purification||Titer Calculations||HPCL-SEC Purity||clEF Purity|
|Sample#||ID||Sample Volume(ml)||Harvest Processed (ml)||Elution Volume (ml)||Conc.by A280 (mg/ml)||Yield of protein A Eluate (mg/ml)||Conc.by Octet (mg/ml)||Load (mg)||(Yield/Load)* 100-Titer Recovery (%)||Main Peak (%)||HMWS (%)||LMWS (%)||Main Peak (%)||Sum of basic (%)||Sum of Acidic (%)||Averagepl|
Downstream and Analytics Summary
5L BR Growth Profile – VCD & Viability
5L BR Growth Profile – Metabolic Profile
5L BR – pH, Gas, & Osmolality
5L BR – Summary Table
|Summary Table for the BioXpress 5L Runs|
|ID||PVCD X106 c/mL||Run Duration Days||Harvest Viability%||Harvest Titer mg/L|
5L BR – Analytics Summary
BioExpress 5L Bioreactor Purification Data
|Protein A Drip Column Purification||Titer Calculations|
|Sample#||ID||Sample Volume(ml)||Harvest Processed (ml)||Elution Volume (ml)||Conc.by A280 (mg/ml)||Yield of protein A Eluate (mg/ml)||Conc.by Octet (mg/ml)||Load (mg)||(Yield/Load)* 100-Titer Recovery (%)|
|HPCL-SEC Purity||clEF Purity|
|Sample#||ID||Main Peak (%)||HMWS (%)||LMWS (%)||Main Peak (%)||Sum of basic (%)||Sum of Acidic (%)||Averagepl|
5L BR Glycan Profile
Glycan profile for 5L BR1 and BR2 improved the glycan profile to be comparable to the innovators.
Case study – Anti-inflammatory Tumor Necrosis Factor Inhibiting Agent using Aragen’s proprietary GS Vector
|Summary Table for Aragen CHO GS Anti-inflammatory Tumor Necrosis Factor Inhibiting Agent MP SFQ|
|MP ID||% Viability at Harvest||PVCD E6 c/mL||Harvest Titer (mg/L)|
|Summary Table for Aragen CHO GS Anti-inflammatory Tumor Necrosis Factor Inhibiting Agent SSC SFQ|
|SCC ID||% Viability at Harvest||PVCD E6 c/mL||Harvest Titer (mg/L)|
Stability study of Single Cell Clones (SCC) from Bulk Pool (BP)
Typical Instruments utilized for: Media and feed screening, process optimization in bioreactor and scalability
To Increase the Titer & Robust scalable USP Development
Drastically improves productivity and enables full DoE experiments to be performed at a fraction of the cost compared with traditional bench top reactors.
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.
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.
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 email@example.com