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Technologies & Platforms

Case study: Developability assessment

Background

Client asked WuXi Biologics to assess the key attributes of two candidate molecules to determine the lead molecule for further product CMC development.

Challenge

Design developability platform, identify key selection criteria and key product attributes to determine the lead clone and molecule.

Solution/Results

  • 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.
Table 1 – Results
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

 

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Case study: Balancing expression level and product quality

Background

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.

Challenge

Achieve client request under an expedited timeline.

Solution/Results

  • 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.
Table 1 – CLD Results
Criteria Result
Titer Pool Titer: 1.5 g/L
Clone Titer: 3.6 g/L
Monoclonality
Documented pre-plating
ViCell pictures and ClonePix
colony pictures

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Case Study: FUT8 KO using CRISPR/Cas9 Technology

Background

Demonstrate reduction of fucosylation levels and increase ADCC activity.

Challenge

Reducing the fucosylation levels to less than 1% (starting levels of parental clone was 91%) and obtain improved ADCC activity by 5X or more in 2-3 month timeframe.

Solution/Results

WuXi utilized CRISPR/Cas9 technology during cell line development to decrease fucosylation levels to desired levels and dramatically increase ADCC activity (see Table 1).

Table 1 – Results
Clones Titer(g/L) Fucosylation Level ADCC
Parental Clone 2.79 91% 154%
Clone 1 2.73 0.9% 889%
Clone 2 2.31 0.4% 1462%

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Case study: Strategies with a shear sensitive clone

Background

  • The initial process had a low growth, high lactate and very low titer of 0.2 g/L.

Challenge

  • The clone was shear sensitive and grew poorly with microsparger.

Solution/Results

  • The WuXi 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

Background

  • The initial process had significantly different glycan profiles, especially G0F, when compared to the reference material.

Challenge

  • G0F >15% is higher than reference material.
  • Titer impact needs to be manageable while making process changes.

Solution/Results

  • 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

Background

  • The initial process had significantly lower ADCC activity when compared to the reference material.

Challenge

  • Dramatically lower ADCC activity (about 50% lower), which could be hard to optimize
  • CDC activity needs to stay within range while optimizing ADCC activity

Solution/Results

  • 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

Background

  • 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.

Challenge

  • 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.

Solution/Results

  • 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

Background

  • 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%.

Challenge

  • Process medium already locked. Can only optimize process conditions

Solution/Results

  • 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

Background

  • Recombinant protein with high demand in productivity and product quality

Challenge

  • Relatively instable product
  • PQA very sensitive to cell culture performance such as viability

Solution/Results

  • 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
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