Background

Client requested a cell line with good titer and product quality.

Challenge

With the third party codon, high titer clones tend to have more aggregates.

Solution

• Used AI codon program for codon optimization
• Evaluated multiple sequences with Transient expression.
• Selected one sequence with higher titer and better product quality for stable cell line development.

Results

• Delivered clones with titer of 4 g/L and SEC main peak above 98%.

Table 1 – CLD Results

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 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.

Background

• The initial process had high afucosylated species with toxicity concern

Challenge

• Afucosylated species >15%, needs to control at below 10%
• Titer impact needs to be manageable while making process changes.

Solution/Results

• Applying a new pH control strategy can successfully decrease afucosylation level.
• No titer compromise under the new pH control strategy.
• Improved process was successfully scaled up to 500L GMP manufacture.

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.

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.

Background

• The process had drastic change in charge variant profile after media change in an effort to reduce CoGs during late phase development. The goal is to optimize the process to match acidic peak species in the original process.

Challenge

• The acidic peak must be reduced by ~10%.

Solution/Results

• A DoE study was executed in spin tube scale down model, where 7-factors were employed.
• The optimized process was confirmed in 3L bioreactors with 10 % acidic peak reduction.

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

Background

WuXi Biologics suggested using an FTE approach to meet critical bispecific antibody development timelines.

Challenge

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.

Solution/Results

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.

Table 3 – FTE program results

Background

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.

Challenge

Develop a high-throughput system that could quickly evaluate over 130 different molecules using multiple analytical methods to meet critical client timelines and budget.

Solution/Results

A well-established, HT matrix-screening method in a fast, efficient way.

Background

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.

Challenge

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.

Background

A client, now under time pressure to meet IND filing milestones, requested an increase in product titer/cell line productivity and better product quality of a difficult to produce bispecific antibody developed at another CRO.

Challenge

After initial technical transfer and review of the existing cell line and product quality data from the client, WuXi Biologics CLD team, along with the client technical team, decided it needed to go back into cell line development and use our WuXia CHO-K1 platform to generate a new clonal cell line and develop a new cell culture process to optimize productivity and product quality attributes. This effort would require expedited CLD and product development timelines.

Solution/Results

After finding the ideal clone coming from transfection and bulk pool screening, we also utilized our high-throughput transient cell culture optimization platform to find the best conditions for stable cell line construction and cell culture conditions. As the result, the final titer of this difficult-to-express bispecific antibody was boosted to 8.4 g/L, and the purity was improved to 95%. It took less than 6 months to make these improvements thus allowing the client to meet their IND filing milestones.

Background

At the start of the pandemic, a call to action to the pharma/biopharma industry was made to quickly develop vaccines and treatments to the COVID-19 virus. Multiple companies responded by discovering several promising neutralizing antibodies to SARS-CoV-2 and due to our true, one-stop, integrated and efficient antibody CMC development platform, these companies requested expedited product development to get their much-needed biologics into the clinic and on to patients in need.

Challenge

To work with our clients and the global regulatory agencies to develop a record-breaking CMC development and GMP manufacturing timelines without compromising product quality and also meet the stringent CMC regulatory requirements.

Solution/Results

In the peer-reviewed articles presented here and here, we report a novel Chemistry, Manufacturing, and Control (CMC) workflow and demonstrate a significantly shortened timeline of 3-6 months from DNA to Investigational New Drug (IND) application for seven different antibody programs. Several of these programs went on to receive Emergency Use Authorization (EUA) approval in less than two years. In summary, due to the vast experience, process understanding and technology platform knowledge of the WuXi Biologics WuXia CHO-K1 cell line development platform, materials generated from stable pools under GMP conditions were used for rapid initiation of phase I clinical trials in approximately 3 months. Clones with comparable product quality as pools were subsequently selected for further development and manufacturing and enabled an IND application filing within 6 months. Stability studies on the critical path were allowed a shorter duration prior to IND filing thus greatly reducing the time required for final clone determination (extended stability data would be provided on a rolling basis to the regulatory authorities) . NGS-based viral testing was routinely used for rapid conditional release of the Master Cell Bank (MCB) for GMP production (with more comprehensive CLC characterization per ICH guidelines provided on a rolling basis to the regulatory authorities). The successful execution of these COVID-19 programs relied on our robust, fit for purpose, and highly-vetted CLD platform.

Background

A client requested a timeline of only 9 months from the initiation of cell line development activities to IND-filing for a novel bispecific nanobody.

Challenge

The tight timeline and the non-mAb format of the molecule put additional strain on the development teams as no previous development platform had been established for bispecific nanobody formats. In cell line development, the significant hurdle facing the development team was a lack of high-throughput screening methods to determine the high-expression clones in microwell plates.

Solution/Results

In rapid fashion, a high-quality customized OCTET method was efficiently developed and validated to ensure the successful screening and determination of high-expression clones. This method also enabled in-process testing of clone expression levels during fed-batch studies. A second challenge was discovered when a common titer decrease was observed from day 11 to day 14 of fed-batch culture. Rapid troubleshooting by the team reported a correlation between end-culture cell viability and titer decrease. With agile cross-team communication and collaboration, process optimization was performed and a drop in culture temperature was found to effectively enhance clone titers and alleviate the titer decrease during the time specified previously. The final clone recorded impressive stability (~4% titer variation) and a 2.6-fold enhancement of expression level.

Background

Sialic acids are terminal, negatively charged monosaccharides present on many N- and O-glycans. The efficacy, serum half-life and immunogenicity of therapeutic antibodies are impacted by both the abundance and the types of sialic acids. A client brought to WuXi Biologics a preferred therapeutic antibody but it did not have the desired sialylation and the CLD team was asked if they could determine mechanisms to increase the amount and appropriate type of sialylation on the molecule.

Challenge

Achieving the desired sialylation without going back into cell line development and performing rescreening of clones is challenging as many factors (e.g., cell line genetics, post-translational modifications and cell culture conditions) impact sialylation. It was determined that multiple glycosylation sites were available for sialylation. The WuXi Biologics team needed to quickly address many factors and determine a path to achieve an increase in sialylation to meet project milestones and timelines.

Solution/Results

Using a QbD approach to look holistically at all factors impacting sialylation was the key for the successful outcome. During a preliminary material generation experiment, it was noticed that the harvested product contained two primary cIEF peaks indicating two different sialylated groups of molecules. The composition of Group A was confirmed as highly sialyated modified species via de-sialyation cIEF, while Group B demonstrated a lesser-sialylated species. Multiple strategies, including modified Fed Batch feeding plan and various additives, were applied to successfully boost the desired Group A%.

Background

A client reached out to WuXi Biologics to redevelop, starting with cell line development, a monoclonal antibody (mAb) that was developed at a third party CDMO. The reason for the redevelopment was due to undesirable levels (>14%) of mannose (Man5) on the molecule, despite the fact that the cell line had other strong quality attributes such as product titer (5.9 g/L).

Challenge

The CLD team was requested to go back into CLD using the WuXi Biologics WuXia CHO-K1 CLD platform to find a stable and high producing clone at or greater than 5.9 g/L but had acceptable mannose levels. Also, the client, having to now redevelop the antibody requested an expedited timeline to try and meet original project timeline milestones.

Solution/Results

Using our proprietary codon optimization platform, we optimized the mAb sequence codon and utilized our WuXia CHO-K1 host cell line to boost the titer to 6.9 g/L in just 2.5 months. The Man5 level was successfully controlled to ~4.4% throughout the extended cell culture duration (seed to completion of Fed-batch large scale production). The higher titer and ideal product quality cell line developed by us helped the client to successfully switch production cell lines for late-stage development.

Background

A gene of interest can exhibit distinct expression levels and product quality attributes in a given host cell line when different codons are employed, which will affect the cost and efficiency of biologics development and manufacturing. Here, we describe the augmenting effect of a custom codon optimization method, based specifically on the codon and codon-pair usage frequencies of our in-house CHO-K1 host cell line, on the expression level of multiple therapeutic antibodies. We show that our custom codon optimization is effective both in CHO-K1 transient and stable expression systems, and improves the productivity of stable cell lines intended for biologics manufacturing.

Challenge

Protein expression levels vary greatly depending on the codons employed in the gene of interest and the host cell lines used.

Solution/Results

Using a codon optimization method, based specifically on the codon and codon-pair usage frequencies, an increase in the expression level of multiple therapeutic antibodies was demonstrated in our in-house CHO-K1 transient and stable expression systems.

Background

For clients with multiple lead molecule candidate, developability is an important consideration when moving into the CMC phase. Attributes such as manufacturability, stability, immunogenicity need to be assessed in order to predict and mitigate the risk of development failure.

Challenge

Provide clients with robust development assessment services to facilitate informed decision on lead molecule selection, reduce CMC failure, save cost and boost timeline.

Solution/Results

Our in silico assessment contains three core-aspects: sequence analysis, aggregation propensity, and immunogenicity. In sequence analysis, the sequence elements are checked and the unknown domains/motifs are identified in order to check for any hot spots potentially susceptible to chemical and enzymatic modifications. For aggregation propensity, properties of a given protein such as surface hydrophobicity, charge, and free-cysteine residues are used to calculate an aggregation probability. Lastly, a molecule’s immunogenicity risk is assessed by computing the sequence binding possibility to MHC molecules and comparing the sequence similarity to the human BCR database.