Case studies for development services
- Developability assessment
- Balancing expression level and product quality
- Strategies with a shear sensitive clone
- Glycan profile optimization to reduce G0F
- ADCC optimization
- Growing NS0 cells in disposable bioreactors
- CEX improvement via DoE
- Steady state perfusion with high cell density and viability
- FTE-Model Approach
- Protein Refolding Analysis using HT-Approach
- Trouble-shooting product issues in GMP manufacturing
Case study: Developability assessment
Client asked WuXi Biologics to assess the key attributes of two candidate molecules to determine the lead molecule for further product CMC development.
Design developability platform, identify key selection criteria and key product attributes to determine the lead clone and molecule.
- Both candidate molecules had the same product pool titer.
- Molecule 1 exhibited aggregation and purification issues and lower solubility.
- Taking all factors into consideration (see examples in Table 1), Molecule 2 was selected for cell line cloning and CMC development.
- After further development and optimization, a titer of 9.7 g/L was achieved and the IND-filing was submitted in April 2016.
|Criteria||Molecule 1||Molecule 2|
|Fit to Upstream||3.7 g/L||3.7 g/L|
|Fit to Downstream
||>10% HMW level after ProA
52% CEX step yield
|87% CEX step yield|
|Formulation and Stability
||Platform Solubility: 100mg/ml||Platform Solubility: >150mg/ml|
|Biophysical and Biochemical Characterization
||DSC: Tm2: 76.4oC||DSC: Tm2: 78.7oC|
Case study: Balancing expression level and product quality
Client requested a cell line with product titer greater than 1.5 g/L while maintaining high product quality. Client also requested assurances of monoclonality for the selected cell clone.
Achieve client request under an expedited timeline.
- Optimized the DNA Sequence of the molecule prior to transfection and utilized a dual selection system for cell line development. Two rounds of cloning (via ClonePix) were conducted to ensure monoclonality.
- Evaluated two different media and two different fed-batch cell culture conditions to optimize titer beyond client expectations (see Table 1 for titer results).
- Delivered cell line per client specified timeline.
|Titer||Pool Titer: 1.5 g/L
Clone Titer: 3.6 g/L
ViCell pictures and ClonePix
Case study: Strategies with a shear sensitive clone
- The initial process had a low growth, high lactate and very low titer of 0.2 g/L.
- The clone was shear sensitive and grew poorly with microsparger.
- The WuXi Biologics team replaced microsparger with drilled-hole sparger & optimized bioreactor conditions.
- We further improved the process by basal & fed-batch media/feed optimization.
- The effort led to a much better cell growth, cell viability, lactate profile and an improved titer of >1.5 g/L.
Case study: Glycan profile optimization to reduce G0F
- The initial process had significantly different glycan profiles, especially G0F, when compared to the reference material.
- G0F >15% is higher than reference material.
- Titer impact needs to be manageable while making process changes.
- Adding medium component X can successfully bring down G0F content.
- Component X led to lower titer while bringing down G0F content.
- Improved process had satisfactory glycan profile and acceptable titer.
Case study: ADCC optimization
- The initial process had significantly lower ADCC activity when compared to the reference material.
- Dramatically lower ADCC activity (about 50% lower), which could be hard to optimize
- CDC activity needs to stay within range while optimizing ADCC activity
- Adding medium components allowed ADCC activity adjustment and without significant impact on CDC activity. Both ADCC and CDC were within target range after optimization.
- Strong correlation between ADCC activity and total a-fucosylated glycan content (G0+G1+G2+Man5).
- Valuable tool for molecules that need ADCC functions.
Case study: Growing NS0 cells in disposable bioreactors
- Direct transfer of process from client to WuXi Biologics for clinical trial production.
- Client required comparability assessment to the legacy manufacturing process and final product.
- No developer of the original process was available. Had to perform only document-based process transfer.
- New process to be conducted in single-use bioreactors while the original process was performed through fixed stainless steel tanks/pipeline.
- Cell culture of NS0 cell line is difficult in single-use disposable systems according to historical records.
- Through extensive PD effort, the manufacturing process was successfully transferred, optimized and performed in the single-use systems.
- Process scale-up in single-use systems was confirmed at 50 L and 250 L scale and likewise GMP manufacturing was conducted at 2,000 L scale in single-use bioreactors.
- Comparability data in IND filing has been accepted by the U.S. FDA.
- The 1st biological drug manufactured in China for global clinical trials was successfully produced by WuXi Biologics’ manufacturing site in Wuxi city.
Case study: CEX improvement via DoE
- The initial process met titer target and had locked in basal and feed medium already. However, client wanted to further improve product quality by reducing acidic variant from ~28% to <15%.
- Process medium already locked. Can only optimize process conditions
- A DOE study was executed, where temp and pH (two main process conditions known to impact charge variant) were main inputs.
- A design space was obtained where titer can be maintained and acidic variant level is reduced to 13%.
Case Study: Steady state perfusion with high cell density and viability
- Recombinant protein with high demand in productivity and product quality
- Relatively instable product
- PQA very sensitive to cell culture performance such as viability
- Specific productivity is limited due to the nature of the protein, high viable cell density is required to achieve high productivity
- ATF system was introduced as cell retention device to achieve high viable cell density; proper cell removal (bleeding) rate was used to maintain stable viable cell density and viability
- Long term steady state perfusion (typically 60 days, up to 130 days) with stable cell growth performance, metabolism, productivity and PQA
- Clarified harvest from ATF enables direct product capture downstream process
- Perfusion process successfully scaled up to 250L (with 150L working volume) GMP runs
Case Study: FTE-Model Approach
WuXi Biologics suggested using an FTE approach to meet critical bispecific antibody development timelines.
Previous client attempts to produce and purify the protein led to undesirable expression levels of a bispecific antibody due to low expression of one of the antibody chains which resulted in only 10% of desired protein product from the entire protein production campaign. These challenges were delaying product CMC development and thus time was critical as original IND filing date was in jeopardy.
After discussion with the client and outlining the DOE study required to potentially solve the problem, client-dedicated personnel via an FTE program was established. The client-dedicated FTE team generated and evaluated 42 different constructs and evaluated expression levels of the bispecific antibody in over 1,000 minipools using a high-throughput methodology. Minipools demonstrating best results were moved to clone screening and eventual final clone selection. Results provided in Table 3.
|Final Titer of bispecific Ab clone||Main product expression levels before purification||Main product purity after purification||Date of client IND filing|
|3.8 g/L||90%||98%||(On-time filing)|
Case Study: Protein Refolding Analysis using HT-Approach
Client struggled with developing the tools to understand if a recombinant cytokine product produced in e. coli had refolded correctly after removal from inclusion bodies post fermentation.
Develop a high-throughput system that could quickly evaluate over 130 different molecules using multiple analytical methods to meet critical client timelines and budget.
A well-established, HT matrix-screening method in a fast, efficient way.
Case Study: Trouble-shooting product issues in GMP manufacturing
Client could not get a consistent product quality or GMP manufacturing process performance at current vendor. Requested WuXi Biologics assemble a team from multiple functional areas to trouble-shoot manufacturing process and provide solutions.
Product had high HMW and dimer content and low enzymatic activity. Had to evaluate over 800 pages of batch records, perform technical transfer and trouble-shoot based on investigation master plan in short time frame.