Current trends and challenges in the downstream purification of bispecific antibodies

Abstract

Bispecific antibodies (bsAbs) represent a highly promising class of biotherapeutic modality. The downstream processing of this class of antibodies is therefore of crucial importance in ensuring that these products can be obtained with high purity and yield. Due to the various fundamental structural similarities between bsAbs and monoclonal antibodies (mAbs), many of the current bsAb downstream purification methodologies are based on the established purification processes of mAbs, where affinity, charge, size, hydrophobicity and mixed-mode-based purification are frequently employed. Nevertheless, the downstream processing of bsAbs presents a unique set of challenges due to the presence of bsAb-specific byproducts, such as mispaired products, undesired fragments and higher levels of aggregates, that are otherwise absent or present in lower levels in mAb cell culture supernatants, thus often requiring the design of additional purification strategies in order to obtain products of high purity. Here, we outline the current major purification methods of bsAbs, highlighting the corresponding solutions that have been proposed to circumvent the unique challenges presented by this class of antibodies, including differential affinity chromatography, sequential affinity chromatography and the use of salt additives and pH gradients or multistep elutions in various modes of purification. Finally, a perspective towards future process development is offered.

Keywords: bispecific antibody; capture chromatography; downstream purification; polishing chromatography; product-related impurities.

Figure 1

(a) Schematic representation of an immunoglobulin G (IgG) monoclonal antibody (mAb), which consists of two heavy chains (HCs, dark green) and two light chains (LCs, light green). The HC comprises of VH, CH1, hinge, CH2 and CH3 domains, whereas the LC comprises of VL and CL domains. The VL, VH, CL and CH1 domains make up the antigen-binding fragment (Fab), whereas the CH2 and CH3 domains constitute the crystallizable fragment (Fc) region. The VH and VL domains make up the variable fragment (Fv) domain. The major affinity ligand-binding sites are also indicated with an arrow at the respective positions on the IgG. (b–d) Schematic representation of certain bsAb formats within the three different groups of bsAbs, namely the asymmetric (b), symmetric (c) and fragment-based bsAbs (d). The valency of each bsAb is indicated in bold and italics below eachbsAb.

Figure 2

Impurities formed as a result of HC mispairing (light blue dotted box), LC mispairing (light red dotted box), fragmentation (light green dotted box) and aggregation (light purple dotted box) are illustrated here for a representative desired asymmetric bsAb, with the proposed strategies to remove them shown in the corresponding dark colored non-dotted boxes. Not shown here: (1) for a Fab × scFv bsAb, differential KappaSelect affinity and CH1-based chromatographic methods have proven to be useful [59, 60]; (2) diabody-IgG mispaired products can be separated from scFv-IgG bsAb targets through the use of an alkaline pH near the pI of the bsAb using cation exchange chromatography [71]; (3) hydrophobic and mixed-mode resins have been proposed to provide good separation for certain homodimer mispaired products from their bsAb targets [77, 78]. Examples listed here are not exhaustive.