Application of Platform Approaches to the Development of Bispecific Antibodies

Finding the Perfect Balance Between Speed and Quality in Bispecific Antibody Production

While monoclonal antibodies (mAbs) readily fit platform approaches to purification and product quality analytics, the variable physicochemical properties of bispecific mAbs (BsAbs) mean that they are prone to fragmentation, formation of homodimers and aggregation. These challenges, coupled with low titer and yield, can ultimately lead to more extensive processes and analytical development when compared to mAbs.

However, provided that the chosen CDMO partner possesses the technical expertise and experience, and based on an evaluation of the molecule’s personality, particularly in cases where the BsAb molecule behaves very similarly to a mAb, the application of platform approaches to the development of bispecific antibodies should be considered. If proven to be viable, such approaches could yield benefits to the client in the form of an accelerated production timeline that encompasses comprehensive product quality assessments.

Whatever the Scenario our Team of Experts are Equipped to Navigate our Clients Through Their Critical Path to Success

FUJIFILM Diosynth Biotechnologies (FDB) has extensive expertise in process development and cGMP manufacture of BsAbs, bi- and tri-specifics, Fc-fusion, Fc regions, ScFv domains and a range of antibody-like conjugates. For cell line development, our robust and versatile Apollo™X mammalian expression system is suitable for the expression of BsAbs, generating headline titers of up to 5 g/L (and 10 g/L for mAbs). Meanwhile, specifically for BsAbs purification, options include either our mAb platform (where determined to be suitable) or custom process development.

Our mAb purification platform has proven itself to be suitable for the purification of proteins both from client-specific cell lines as well as from Apollo™X. In this scenario, we describe the purification of a BsAb generated outside of our Apollo™X platform and the application of our mAb platform purification approach (benefits of this approach are outlined in Figure 1).

Figure 1: Benefits of FDB’s mAb Platform Approach

• Fully single-use platform: Improved speed-to-market and greater flexibility for process development at the necessary scale for future commercial success
• Rapid implementation: Standardized bill of materials, platform process and analytical methods
• Prompt start: Off-the-shelf consumables and manufacturing slots allocated to early phase clients allowing for a prompt start
• Accelerated timelines: Toxicology material supply at 8 months and drug product at 12 to 13 months from start of cell line development
• Expertise and experience: Dedicated team of experts with experience of 30+ programs. Our staff are part of expert groups shaping the future of the industry
• Go further: As your Partner For Life, we offer in-house scale out, scale up, and continuous manufacturing solutions for late phase and commercial supply

Applying Bioinformatics to Determine the Suitability of our Platform Approach to the Development of a BsAb

As the developability of BsAbs cannot be predicted from their parent antibodies, we apply bioinformatics in combination with small scale runs to determine whether any novel antibody format is a suitable candidate for processing and purification using our platform approach. Bioinformatics analysis can be used to predict the surface exposed hydrophobic, positive and negative patches that fujifilmdiosynth.com can lead to aggregation, identify potential chemical liabilities including oxidation and deamidation sites and determine the isoelectric point (pI) of the conjugated form of the molecule. Taken together, these early insights mean our experts can assess our workflows before purification even begins.

After performing this early bioinformatic assessment, we determined that our mAb platform approach could be suitable for the expression of a BsAb containing an IgG like Fc region. Here, we describe how only one unit operation in the downstream platform required adaptation to purify this BsAb successfully. Subsequent manufacture following scale up to 2000 L at our College Station, Texas (USA), facility showed comparable product quality compared to the non-GMP 200 L scale run at our Billingham (UK) facility.

Process Development Methods and Results

Our Billingham (UK) facility is equipped to perform 200 L non-GMP runs, with standard procedures for processes transfer to our College Station, Texas (USA) facility for scale up to 2000 L cGMP.

Upstream Process

We implemented our standard transfer and scale up approach for a BsAb, whereby pre-defined growth medium, production supplements and incubator settings were adopted for both non-GMP and cGMP processes. For both scales, comparable levels of growth were observed in the N-1 seed bioreactor with viabilities in excess of 95%. During the production bioreactor phases, peak cell densities greater than 20×106 cells/mL were observed with viability in excess of 85% at the end of production phases. Importantly, both scales achieved comparable titers (5 g/L for 200 L non-GMP run and 6 g/L for 2000 L cGMP run). At the end of production, cultures were harvested through a standard depth filtration set-up, yielding >90% step recovery at both scales.

Downstream Process

Following an initial small-scale run, our mAb platform standard down-stream purification conditions were identified as being appropriate throughout process development and cGMP manufacture, except for an adapted CEX gradient that was needed to achieve optional separation of high molecular weight (HMW) species. Bioinformatics assessments showed that this BsAb contained a relatively high content of hydrophobic patches in both the Fc and Fv domains, predicting the formation of higher order species that were detected in both the Harvested Cell Culture Fluid and Protein A eluate (5-10% HMW). To ensure standard operating CEX parameters performed robustly, our approach for any antibody is to perform a DoE using a scale-down model, this revealed that a shallower gradient slope and UV gate adjustment (to resolve and exclude HMW from monomer species) coupled with increasing the platform centreline target loading achieved the best separation (HMW were reduced from 5-10% to <1.5%). fujifilmdiosynth.com

Analytical Approach

Our standard approach is to initially evaluate platform analytical methods alongside integrated DoE parameters. These include SEC-UPLC (23 DoE conditions varying pH, buffer and salt concentration), Non reducing CE-SDS (23 DoE conditions varying temperature, time and pH) and icIEF (27 DoE conditions varying pharmalyte composition and focussing time). Both SEC-UPLC and Non reducing CE-SDS platform methodologies were found to provide optimal resolution and subsequent qualification demonstrated precision, reproducibility, range, linearity, LOD/LOQ, specificity and accuracy to be acceptable, ensuring system suitability and sample acceptance criteria within the method to be appropriate for cGMP testing. Experience tells us that icIEF often requires a DoE evaluation of pharmalyte composition, focussing time and the use of urea during sample preparation. For this specific BsAb, these conditions were altered to create a product specific methodology that was qualifiable and suitable for cGMP release testing. Method refinement at FDB is performed typically using independently produced reference material. Where this is not available, we provide the option of using CLD material.

Speed to Clinic

The entire technology transfer to FDB and process development phase, including master cell bank creation and 200 L non GMP run, was completed within 8 months. Within 10 months of technology transfer into FDB process specifications were defined. By routine early engagement of process development with manufacturing, we ensure that all process adaptations are well controlled ensuing successful manufacture at scale.

Table 1. Comparison of Product Quality Results at 200 L and 2000 L

Conclusion

Our experts are well versed in establishing antibody products within pre-defined platform guard rails, bringing successful clinical and commercial product to our clients. While platform approaches offer clear benefits in terms of both time and efficiency savings, for novel antibody formats they are not always feasible. Here, we describe how our experts were able to adjust our mAb production and purification platform to be applicable for the rapid development and scale up of a BsAb. For molecules lacking an Fc region that cannot be adapted into our mAb platform, we offer resin screening and process development services. During initial commercial discussions you can expect our experts to navigate you through options that best meet your requirements and de-risk your path to market.

Sam Maurice is a Down-Stream Team Leader at FUJIFILM Diosynth Biotechnologies, UK

Carl Capps is an Up-Stream Principal Scientist at FUJIFILM Diosynth Biotechnologies, UK

Shaun Lawson is an Analytical Principal Scientist at FUJIFILM Diosynth Biotechnologies, UK

Dan Maltman is a Process Transfer Lead at FUJIFILM Diosynth Biotechnologies, UK

Carl Grant is a Process Transfer Lead at FUJIFILM Diosynth Biotechnologies, UK

Rebecca Abram is a Director of Strategic Technical Marketing at FUJIFILM Diosynth Biotechnologies, UK

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