Brian Kelley

Industrialization of mAb production technology: The bioprocessing industry at a crossroads

Manufacturing processes for therapeutic monoclonal antibodies (mAbs) have evolved tremendously since the first licensed mAb product (OKT3) in 1986. The rapid growth in product demand for mAbs triggered parallel efforts to increase production capacity through construction of large bulk manufacturing plants as well as improvements in cell culture processes to raise product titers. This combination has led to an excess of manufacturing capacity, and together with improvements in conventional purification technologies, promises nearly unlimited production capacity in the foreseeable future. The increase in titers has also led to a marked reduction in production costs, which could then become a relatively small fraction of sales price for future products which are sold at prices at or near current levels. The reduction of capacity and cost pressures for current state-of-the-art bulk production processes may shift the focus of process development efforts and have important implications for both plant design and product development strategies for both biopharmaceutical and contract manufacturing companies.

Very large scale monoclonal antibody purification : The case for conventional unit operations

Technology development initiatives targeted for monoclonal antibody purification may be motivated by manufacturing limitations and are often aimed at solving current and future process bottlenecks. A subject under debate in many biotechnology companies is whether conventional unit operations such as chromatography will eventually become limiting for the production of recombinant protein therapeutics. An evaluation of the potential limitations of process chromatography and filtration using todayapos;s commercially available resins and membranes was conducted for a conceptual process scaled to produce 10 tons of monoclonal antibody per year from a single manufacturing plant, a scale representing one of the worldapos;s largest single‐plant capacities for cGMP protein production. The process employs a simple, efficient purification train using only two chromatographic and two ultrafiltration steps, modeled after a platform antibody purification train that has generated 10 kg batches in clinical production. Based on analyses of cost of goods and the production capacity of this very large scale purification process, it is unlikely that non‐conventional downstream unit operations would be needed to replace conventional chromatographic and filtration separation steps, at least for recombinant antibodies.

Exploration of overloaded cation exchange chromatography for monoclonal antibody purification

Cation exchange chromatography using conventional resins, having either diffusive or perfusive flow paths, operated in bind-elute mode has been commonly employed in monoclonal antibody (MAb) purification processes. In this study, the performance of diffusive and perfusive cation exchange resins (SP-Sepharose FF (SPSFF) and Poros 50HS) and a convective cation exchange membrane (Mustang S) and monolith (SO(3) Monolith) were compared. All matrices were utilized in an isocratic state under typical binding conditions with an antibody load of up to 1000 g/L of chromatographic matrix. The dynamic binding capacity of the cation exchange resins is typically below 100 g/L resin, so they were loaded beyond the point of anticipated MAb break through. All of the matrices performed similarly in that they effectively retained host cell protein and DNA during the loading and wash steps, while antibody flowed through each matrix after its dynamic binding capacity was reached. The matrices differed, though, in that conventional diffusive and perfusive chromatographic resins (SPSFF and Poros 50HS) demonstrated a higher binding capacity for high molecular weight species (HMW) than convective flow matrices (membrane and monolith); Poros 50HS displayed the highest HMW binding capacity. Further exploration of the conventional chromatographic resins in an isocratic overloaded mode demonstrated that the impurity binding capacity was well maintained on Poros 50HS, but not on SPSFF, when the operating flow rate was as high as 36 column volumes per hour. Host cell protein and HMW removal by Poros 50HS was affected by altering the loading conductivity. A higher percentage of host cell protein removal was achieved at a low conductivity of 3 mS/cm. HMW binding capacity was optimized at 5 mS/cm. Our data from runs on Poros 50HS resin also showed that leached protein A and cell culture additive such as gentamicin were able to be removed under the isocratic overloaded condition. Lastly, a MAb purification process employing protein A affinity chromatography, isocratic overloaded cation exchange chromatography using Poros 50HS and anion exchange chromatography using QSFF in flow through mode was compared with the MAbs commercial manufacturing process, which consisted of protein A affinity chromatography, cation exchange chromatography using SPSFF in bind-elute mode and anion exchange chromatography using QSFF in flow through mode. Comparable step yield and impurity clearance were obtained by the two processes.

Effect of protein and solution properties on the donnan effect during the ultrafiltration of proteins

Formulation of protein biopharmaceuticals as highly concentrated liquids can improve the drug substance storage and supply chain, improve the target product profile, and allow greater flexibility in dosing methods. The Donnan effect can cause a large offset in pH from the target value established with the diafiltration buffer during the concentration and diafiltration of charged proteins with ultrafiltration membranes. For neutral formulations, the pH will typically increase above the diafiltration buffer pH for basic monoclonal antibodies and decline below the diafiltration buffer pH for acidic Fc‐fusion proteins. In this study, new equations for the Donnan effect during the diafiltration and concentration of proteins in solutions containing monovalent and divalent ions were derived. The new Donnan models obey mass conservation laws, account for the buffering capacity of proteins, and account for protein‐ion binding. Data for the pH offsets of an Fc‐fusion protein and a monoclonal antibody were predicted in both monovalent and divalent buffers using these equations. To compensate for the pH offset caused by the Donnan effect, diafiltration buffers with pH and excipient values offset from the ultrafiltrate pool specifications can be used. The Donnan offset observed during the concentration of an acidic Fc‐fusion protein was mitigated by operating at low temperature. It is important to account for the Donnan effect during preformulation studies. The excipients levels in an ultrafiltration pool may differ from the levels in a protein solution obtained by adding buffers into concentrated protein solutions due to the Donnan effect.

Establishing a control system using QbD principles.

Quality by design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody. This chapter describes the elements and tools used to establish acceptance criteria and an attribute testing strategy (ATS) for product variants and process related impurities. The acceptable ranges for CQAs are set based on their potential impact on efficacy and safety/immunogenicity. This approach is focused on the management of patient impacts, rather than simply maintaining a consistent analytical profile. The ATS tools were designed to identify quality attributes that required process and/or testing controls, or that could be captured in a monitoring system to enable lifecycle management of the control strategy.

Integration of QbD risk assessment tools and overall risk management

Quality by design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody. This chapter describes how the risk assessments, logic and interactions of the tools are designed to connect the set of QbD tools and elements into an overarching risk management system. The tools allow comparisons of options based on elective decisions that the sponsor could take and reflect relative values of these options. The overall risk management strategy assures product quality from this enhanced set of assessments and employs a science and risk based approach resulting in a consistent and transparent set of process and product controls and a rational monitoring system.

The rapid identification of elution conditions for therapeutic antibodies from cation-exchange chromatography resins using high-throughput screening

Cation-exchange chromatography is widely used in the purification of therapeutic antibodies, wherein parameters such as elution pH and counterion concentration require optimization for individual antibodies across different chromatography resins. With a growing number of antibodies in clinical trials and the pressure to expedite process development, we developed and automated a high-throughput batch-binding screen to more efficiently optimize elution conditions for cation-exchange chromatography resins. The screen maps the binding behavior of antibodies and impurities as a function of pH and counterion concentration in terms of a partition coefficient (Kp). Using this approach, the binding behavior of a library of antibodies was assessed on Poros 50HS and SP Sepharose Fast Flow resins. The diversity in binding behavior between antibodies and across resins translated to the requirement of a variable counterion concentration to elute each antibody. This requirement can be met through the use of a gradient elution. However, a gradient of increasing counterion concentration spans the transition from binding to non-binding for impurities as well as the antibody, resulting in the elution of impurities within the antibody elution peak. Step elution conditions that selectively elute the antibody while retaining impurities on the resin can now be rapidly identified using our high-throughput approach. We demonstrate that by correlating antibody Kp to elution pool volume and yield on packed-bed columns and through the calculation of a separation factor, we can efficiently optimize step elution conditions that maximize impurity clearance and yield for each antibody.

Quality by Design risk assessments supporting approved antibody products

Quality by Design (QbD) is a global regulatory initiative that enhances pharmaceutical development through the design of the manufacturing process and controls to consistently deliver a product that performs as intended. The principles of pharmaceutical development relevant to QbD are described in the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidance documents ICHQ8-11. In 2008, the Food and Drug Administration (FDA) initiated a QbD pilot program for biological products wherein companies could contribute either with full biological license applications or supplements. Many biopharmaceutical firms participated in the pilot program, and progress was made on establishing the basis for preand post-approval filings. One outcome of the pilot was an approval, granted in 2010, for an expanded change protocol for multiproduct/multisite transfer of drug substance processes for production of monoclonal antibodies at Roche/Genentech. The A-mAb case study provided another substantial contribution to the field. It described a variety of approaches to the major elements of QbD used by 7 companies (Pfizer, GlaxoSmithKline, Genentech, Abbott, Amgen, Lilly, MedImmune) with experience in the development and commercialization of biologics. This publication presented a diverse set of solutions to common problems, but, because many companies were involved, it lacked a self-consistent formalism. Advances in refining the applications of QbD principles have continued in recent years, although progress is slower than hoped. Roche/Genentech has licensed 2 therapeutic recombinant monoclonal antibody products, obinutuzumab (Gazyva ) and atezolizumab (Tencentriq ), in the US using QbD principals. We believe these represent the first approvals for biologics that were comprehensively based on QbD information, including approved design space claims as well as a post-approval lifecycle management plans, contained in the license application. Roche/Genentech recently published 7 articles describing the application of the principles of QbD for development and licensure of therapeutic monoclonal antibodies. These articles present a self-consistent set of risk assessments and logical elements developed over the last decade, based on refinement through the FDA and European Medicines Agency pilot QbD programs and approvals of obinutuzumab and atezolizumab. They provide a standardized basis for the global health authorities to assess new product license applications from Roche/Genentech, and seek to establish transparent communication of the links between the manufacturing process and product storage, product quality and impact to patients, the commercial control strategy, and post-licensure change management. The articles cover all key elements of QbD, including establishment of critical quality attributes, definition of a design space, identification of critical process parameters, assembly of the commercial control system, description of the postapproval life cycle management, considerations of an overarching risk assessment that addresses elective decisions taken at the time of license application, and analysis of how the full set of assessments manage the residual risk to product quality. The refinement of these tools benefitted from the substantial experience the company has gained in over 25 years of biological drug development, which included licensure and production of 9 commercial monoclonal antibodies and the use of applicable process and product platform knowledge. Roche/Genentech view these articles as an open-source sharing of the results of a decade-long internal investment. We hope that the tools will be used by other companies (adapted to their particular product requirements, if needed), which could advance the adoption of improved methodologies in support of license applications for products with enhanced product and process knowledge. This formalism will be used for all biologics in the Roche/Genentech pipeline, and, when combined with the use of process and product platform knowledge, should result in significant efficiencies and resource savings during late-stage development. We also hope that this will enable postlicensure flexibility for production and the appropriate regulatory oversight for process parameter changes within the design space. The large commercial and clinical biologics portfolio at Roche/Genentech will build on this QbD foundation for both life-cycle management and the commercialization phase. Although discussed in the context of antibody products, the tools we describe in the articles should be applicable to other protein therapeutics, and perhaps to small molecules and other pharmaceutical modalities as well. We encourage publication of future refinements of these tools and advances

Post-Licensure Purification Process Improvements for Therapeutic Antibodies: Current and Future States

Abstract Therapeutic antibodies have been manufactured since the advent of intravenous immunoglobulin in the 1950s. Rituxan was the first recombinant monoclonal antibody approved for therapeutic use by the U.S. Food and Drug Administration (FDA) in 1997. Now, over 50 recombinant monoclonal antibodies have been licensed for commercial distribution [ 1 ]. Post-licensure manufacturing process changes provide opportunities to increase manufacturing capacity, improve product quality, consistency, or safety, and may be necessary upon process transfer to a new facility [ 2 ]. Yet, the complexity of global regulatory authority oversight and approval sets a high bar for manufacturing process changes for an approved parenteral protein therapeutic. This review examines the history of antibody production, focusing on purification process technology. Strategies for post-licensure changes are discussed, including regulatory considerations. Case studies are described for several antibody process purification changes. Finally, some speculations are shared about the potential future of purification process technology and post-licensure process changes for commercial therapeutic antibody production.

The Impact of High-Titer Feedstreams on Monoclonal Antibody Purification

Process development groups defining purification processes for therapeutic monoclonal antibodies are finding themselves at a crossroads. High titer feedstocks have challenged processes and facilities to recover single batches in excess of 50 kg, and triggered debates regarding the future direction of mAb purification. Are novel unit operations required to handle these high productivity processes in a cost-effective manner, or will the new generation of resins and membranes be sufficient? This workshop reviewed the state-of-the art in cGMP antibody purification processes, economics and cost of goods considerations, and facility fit issues surrounding novel as well as conventional purification technologies. In addition, pressures to adopt a mAb processing platform continue to mount, most recently through the introduction of QbD and an increasing frequency of multi-product facilities switching between legacy and newly launched mAbs. As modern processes become more industrialized (by convergence on a common set of highly-efficient, scalable, cost-effective unit operations across the industry), development strategies will need to be refined. If further advances in mAb bioprocessing are needed, where should companies focus their technology development efforts for downstream processing?