Bispecific antibodies (bsAbs) are antibodies that can bind two different epitopes or targets simultaneously, enhancing the selectivity and functional affinity of antibodies, thus improving the efficacy of drugs (Labrijn, Janmaat et al. 2019, Li 2019, Li, Wang et al. 2020). As a new type of antibody drugs, bispecific antibodies are a trending area in pharmaceutical research and development. However, there are still many challenges, such as low yield of antibody products, complex purification process, and difficulty in clinical translation. With the continuous advancement of protein engineering technology, the types of bispecific structures are becoming more diverse, where heavy and light chains are combined in various quantities and form to a variety of molecular structures (Kontermann and Brinkmann 2015). However, the diverse molecular structure of bispecific antibodies leads to the production of various byproducts, posing a challenge in their downstream removal.
Currently, bsAbs can be divided into two main categories based on their structure: Non-IgG like and IgG-like(Zhang, Li et al. 2017). Most of the non-IgG like molecules lack the Fc domain. The main advantage of non-IgG like molecules is their small molecular weight, which allows for expression in prokaryotes to reduce costs and enables better tissue penetration to reach tumor sites. However, the lack of an Fc functional region also results in a short half-life in vivo, necessitating continuous administration to sustain the therapeutic effect (Brinkmann and Kontermann 2017). Due to the absence of an Fc domain, non-IgG-like molecules cannot be captured by protein A resin. This increases the complexity in the removal of HCP, for the separation of HCP and target protein was achieved traditionally through the affinity between target protein and protein A, calling for an innovative strategy to remove HCP in the target bsAb. IgG-like with Fc domain can be further divided into two types: symmetry and asymmetry, the majority of bispecific IgG molecules are asymmetric, while IgG fusion proteins often are symmetric in their molecular composition (Brinkmann and Kontermann 2017, Li, Wang et al. 2020). In general, asymmetry involves two different heavy chains and two different light chains(Chen and Zhang 2021), due to the random assembly of the chains, misassembled species could account for approximately 90% of the total mass (Guo, Han et al. 2020, Li, Wang et al. 2020). Homodimers have similar physicochemical properties to intact antibodies, making it challenging to separate the homodimer from the target product.
To reduce the formation of byproducts, there are various strategies to promote the correct pairing of chains, including knob-into-hole, CrossMab, and DuetMab, and etc. The knob-into-hole method is the most classical and widely applied among them (Chen, Hoi et al. 2022). A knob is generated in one CH3 domain by replacing a small amino acid with a larger one and a hole is generated in the other CH3 domain by replacing a large residue with a smaller one(Chen, Han et al. 2019, Li 2021). Although it can effectively reduce the homodimer, it cannot eliminate the homodimer completely. There are still challenges existing for downstream processes.
At present, the most commonly used purification strategy involves affinity chromatography, followed by two polishing steps. Aggregates and homodimers are primarily removed through affinity and cation exchange chromatography. However, there are still some drawbacks of using affinity chromatography to capture the target molecules. Firstly, affinity purification requires elution at a low pH, which can lead to the formation of aggregates due to the higher aggregation propensity(Manikwar, Mulagapati et al. 2020). Secondly, there is ligand shedding with affinity capture, which can introduce shedding ligands such as protein A. Finally, the industrial application of affinity capture in GMP manufacture is rather limited due to the high cost of affinity resins as well as the difficulty in microbial control because of the poor NaOH tolerance of affinity resins (0.1 M) (Maria, Joucla et al. 2015, Kateja, Kumar et al. 2018). So far, many studies have attempted to find alternative technologies for capturing antibodies. Multimodal or mixed-mode chromatography has recently emerged as a promising candidate for innovative methods of antibody purification. Tsutomu Arakawa et al. used Capto MMC to capture the antibody. In another study, they demonstrated that these new resins can not only capture mAb directly from CHO supernatant obtained after cell culture but also significantly improve the efficiency of the purification process as a whole. Pezzini et al. showed that mixed mode resins can be used as a capture step for antibody purification from a crude culture supernatant of CHO cells. In all cases, the yield and purities were comparable with an affinity chromatography step (Pezzini, Joucla et al. 2011, Arakawa, Kurosawa et al. 2016).
In this study, we utilized a two-step chromatography method to purify the bsAb with a knob-into-hole design. In general, the charges and hydrophobicity of homodimers differ from those of heterodimers because homodimers are less well folded(Chen, Han et al. 2019). Based on the differences, we utilized Capto Adhere instead of affinity chromatography to capture the target protein. We aimed to remove the major byproducts, including aggregates, fragments and homodimers. Our data suggested that Capto Adhere effectively removed the aggregates, fragments and hole-hole dimer. We also found that apart from flow through mode reported by previous study, it can also effectively remove HCP by bind-elution mode (Sakhnini, Pedersen et al. 2019). To further improve the SEC-HPLC and RP-HPLC purity of the target bsAb while removing HCP, AEX with flow through mode was applied as a subsequent polishing step. AEX flow-through mode not only can remove HCD, HCP and viruses, but also can improve purity. However, by optimizing pH and conductivity and utilizing the weak binding mode for AEX, not only is HCP effectively removed, but the product's purity is also improved (D. Kelley, Tobler et al. 2008). In addition, we exploited the slight difference in the isoelectric point between homodimers and heterdimer to remove the hole-hole dimer by optimizing the loading conditions, based on the charge disparity between the hole-hole dimer and bsAb. Therefore, the pH value is maintained between the isoelectric points of the hole-hole dimer and bsAb, which facilitates the elimination of dimers. A higher pH value is advantageous for the removal of HCP (pH 7.8 ± 0.1 and conductivity ≤ 5 mS/cm). Our data demonstrated that HCP and homodimers and aggregates removal, and increased the purity of target protein. The purities of SEC-HPLC and RP-HPLC both improved to 98%, and the residual HCP is less than 10 ppm. Due to the presence of only a two-step chromatography process, which also lacks protein A affinity chromatography, apart from improving purity, the cost can be reduced and the yield can be higher. The procedure described in this study can serve as a reference for removing homodimers from bsAbs.