Here, we will consider three broad categories of bsAbs based on their structures, namely (i) asymmetric, (ii) symmetric and (iii) fragment-based bsAbs ( Fig. KEY STRUCTURAL PROPERTIES OF bsAbs AND THEIR MAJOR BYPRODUCTS To this end, this review aims to present the key structural properties of bsAbs and their associated byproducts, outlining the current major purification methods of bsAbs and highlighting the corresponding solutions that have been proposed to circumvent the challenges, as well as to offer a perspective towards future process development. 1) as well as the presence of bsAb-related byproducts, an understanding of which will aid in the identification of potential challenges and therefore design of the optimal strategy for their downstream processing. Although the optimized downstream processing protocols of mAbs serve as a good starting point for the purification of bsAbs, further optimization cannot be fully eliminated due to the differences in their intrinsic structural and concomitant physicochemical properties ( Fig. Many of the current downstream processing methods of bsAbs are built upon the established purification methods of monoclonal antibodies (mAbs), as there are undoubtedly several structural similarities between these antibodies, with the former being derived from at least parts of the latter ( Fig. Yet, in comparison with the numerous detailed reviews outlining the various different formats of bsAbs, along with the associated upstream platform technologies to generate them in order to minimize product-related impurities and their corresponding therapeutic applications, the review of downstream purification of this important class of antibodies is comparatively limited, which may at least in part be attributed to the fewer publications that focus on the purification of these antibodies. The enormous therapeutic potential of bsAbs has led to the development of over 50 different formats of recombinant bsAbs reported so far. Finally, a perspective towards future process development is offered.īispecific antibodies (bsAbs) demonstrate novel functionalities that yield remarkable promise in improving the drug therapeutic efficacy through the recognition and targeting of two different antigens. 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. 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. 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. 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. Bispecific antibodies (bsAbs) represent a highly promising class of biotherapeutic modality.
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