Bispecific antibodies, while showing great therapeutic potential, pose formidable challenges with respect to their assembly, stability, immunogenicity, and pharmacodynamics. Here we describe a novel class of bispecific antibodies with native human immunoglobulin format. The design exploits differences in the affinities of the immunoglobulin isotypes for Protein A, allowing efficient large-scale purification. Using this format, we generated a bispecific antibody, REGN1979, targeting the B cell marker, CD20, and the CD3 component of the T cell receptor, which triggers redirected killing of B cells. In mice, this antibody prevented growth of B cell tumors and also caused regression of large established tumors. In cynomolgus monkeys, low doses of REGN1979 caused prolonged depletion of B cells in peripheral blood with a serum half-life of approximately 14 days. Further, the antibody induced a deeper depletion of B cells in lymphoid organs than rituximab. This format has broad applicability for development of clinical bispecific antibodies.

There is strong interest in the design of bispecific monoclonal antibodies (bsAbs) that can simultaneously bind 2 distinct targets or epitopes to achieve novel mechanisms of action and efficacy. Multiple bispecific formats have been proposed and are currently under development. Regeneron's bispecific technology is based upon a standard fully human IgG antibody in order to minimize immunogenicity and improve the pharmacokinetic profile. A single common light chain and 2 distinct heavy chains combine to form the bispecific molecule. One of the heavy chains contains a chimeric Fc sequence form (called Fc*) that ablates binding to Protein A via the constant region. As a result of co-expression of the 2 heavy chains and the common light chain, 3 products are created, 2 of which are homodimeric for the heavy chains and one that is the desired heterodimeric bispecific product. The Fc* sequence allows selective purification of the FcFc* bispecific product on commercially available affinity columns, due to intermediate binding affinity for Protein A compared to the high avidity FcFc heavy chain homodimer, or the weakly binding Fc*Fc* homodimer. This platform requires the use of Protein A chromatography in both a capture and polishing modality. Several challenges, including variable region Protein A binding, resin selection, selective elution optimization, and impacts upon subsequent non-affinity downstream unit operations, were addressed to create a robust and selective manufacturing process.

The work presented here describes an ultra scale-down (USD) methodology for predicting centrifugal clarification performance in the case of high cell density fermentation broths. Existing USD approaches generated for dilute systems led to a 5- to 10-fold overprediction of clarification performance when applied to such high cell density feeds. This is due to increased interparticle forces, leading to effects such as aggregation, flocculation, or even blanket sedimentation, occurring in the low shear environment of a laboratory centrifuge, which will not be apparent in the settling region of a continuous-flow industrial centrifuge. A USD methodology was created based upon the dilution of high solids feed material to approximately 2% wet wt/vol prior to the application of the clarification test. At this level of dilution cell-cell interactions are minimal. The dilution alters the level of hindered settling in the feed suspensions, and so mathematical corrections are applied to the resultant clarification curves to mimic the original feed accurately. The methodology was successfully verified: corrected USD curves accurately predicted pilot-scale clarification performance of high cell density broths of Saccharomyces cerevisiae and Escherichia coli cells. The USD method allows for the rapid prediction of large-scale clarification of high solids density material using millilitre quantities of feed. The advantages of this method to the biochemical engineer, such as the enabling of rapid process design and scale-up, are discussed.

In recent years, there has been a strong interest in the development and production of gene therapy products, especially those utilizing adeno‐associated virus (AAV) particles. This is evident with the growing number of clinical successes and agency approvals for AAV therapeutics. Due to this increased investment in this technology, a need exists for scalable commercial production methods to ensure adequate product supply as research in AAV shifts from bench‐scale development to clinical production. The purpose of this review is to summarize current scalable purification techniques that can be employed during the commercial manufacturing of AAV as well as highlight certain development considerations, such as adventitious agent removal and process development using the principals of quality by design.