Manufacturability and functionality assessment of different formats of T-cell engaging bispecific antibodies.

T-cell-engaging bispecific antibodies (T-bsAbs) are promising immunotherapies for cancer treatment due to their capability of redirecting T-cells toward destroying tumor cells. Numerous T-bsAb formats have been developed, each with advantages and disadvantages in terms of developability, immunogenicity, effector functions, and pharmacokinetics. Here, we systematically compared T-bsAbs produced using eight different formats, evaluating the effect of molecular design of T-bsAbs on their manufacturability and functionality. These eight T-bsAb formats were constructed using antigen-binding fragments (Fabs) and single-chain variable fragments (scFvs) of antibodies linked to the crystallizable fragment (Fc) domain of immunoglobulin G. To ensure a fair comparison of growth and production data, we used recombinase-mediated cassette exchange technology to generate the T-bsAb-producing CHO cell lines. The produced T-bsAbs were assessed for their purification profile and recovery, binding capability, and biological activities. Our findings indicated that the manufacturability of bsAbs was adversely affected with increased number of scFv building blocks, while the functionality was affected by the combination of multiple factors, including the binding affinity and avidity of targeting moieties and the flexibility and geometry of formats. These results provide valuable insights into the impact of the format design on the optimal production and function of T-bsAbs.

Leveraging an advanced simulated moving bed approach to achieve 3-component separation for enhanced impurity removal in a non-affinity cation exchange capture step.

One of the key challenges in downstream bioprocessing is to obtain products of high purity in a productive fashion through the effective removal of process and product related impurities. While a classical simulated moving bed (SMB) system operation can typically achieve a 2-component separation between the weakly bound impurities and target species, here we present an advanced SMB approach that can achieve a 3-component separation, including the removal of the strongly bound impurities from the target species. As a proof-of-concept, we demonstrate the enhanced removal of strongly bound host cell proteins (HCP) from the target monoclonal antibody (mAb) through the utilisation of the advanced SMB approach in a non-affinity cation exchange (CEX) capture step. In this way, 1 less polishing step was required to achieve the therapeutic requirements of < 100 ppm HCP and the overall process recovery was increased by ~ 6% compared to the corresponding process that utilised a batch CEX operation. The non-affinity CEX capture platform technology established through the utilisation of the advanced SMB approach presented here can potentially be further applied to address the downstream processing challenges presented by other challenging biotherapeutic modalities to yield a final target product with improved purity and recovery.

Harnessing ceramic hydroxyapatite as an effective polishing strategy to remove product- and process-related impurities in bispecific antibody purification.

Bispecific antibody (bsAb), a novel therapeutic modality, provides excellent treatment efficacy, yet poses numerous challenges to downstream process development, which are mainly due to the intricate diversity of bsAb structures and impurity profiles. Ceramic hydroxyapatite (CHT), a mixed-mode medium, allows proteins to interact with its calcium sites (C-sites) through metal affinity and/or its phosphate sites (P-sites) through cation exchange interactions. This dual-binding capability potentially offers unique bind and elute behaviours for different proteins of interest, resulting in optimal product purity when suitable elution conditions are employed. In this study, the effectiveness of CHT as a polishing step for bsAb purification was investigated across three model molecules and benchmarked against the traditional cation exchange chromatography (CEX). For both asymmetric and symmetric IgG-like bsAb post Protein A eluates, at least 97% product purity was achieved after CHT polishing. CHT delivered a superior aggregate clearance to CEX, resulting in low high molecular weight (HMW) impurities (0.5%) and low process-related impurities in the product pools. Moreover, CHT significantly mitigated "chromatography-induced aggregation" whereas eightfold more HMW was generated by CEX. This study illustrated the developability of CHT in effectively eliminating low molecular weight (LMW) impurities through post-load-wash (PLW) optimization, resulting in an additional reduction of up to 48% in LMW impurities. A mechanistic explanation regarding the performance of impurity removal and mitigation of the chromatography-induced aggregation by CHT was proposed, illustrating unique CHT capability is potentially driven by C-site cooperation, of which effectiveness could depend on the bsAb composition and size.

Development of a T cell-redirecting bispecific antibody targeting B-cell maturation antigen for the suppression of multiple myeloma cell growth.

Background: Multiple myeloma (MM) is the second most common hematological malignancy. It has emerged as one of the next possible hematological diseases amenable to immunotherapy. B-cell maturation antigen (BCMA), a member of the tumor necrosis factor receptor superfamily, is highly expressed in MM cells and is one target with the most potential for developing MM-targeting immunotherapy. Other than the FDA-approved BCMA-targeting CAR T-cell therapy, such as Abecma and CARVYKTI, T cell-engaging multi-specific antibody is another promising therapeutic modality for BCMA-targeting MM treatment. We develop a T-cell redirecting BCMA-targeting bispecific antibody (bsAb) and evaluate its anti-MM activity. Methods: We first generated several clones of mouse anti-human BCMA monoclonal antibodies using DNA immunization. One of the anti-BCMA antibodies was then used to design and produce a T cell-redirecting BCMA × CD3 bsAb in CHO cells. Finally, we examined the effect of the bsAb on MM cell growth both in vitro and in vivo. Results: The BCMA × CD3 bsAb was designed in a FabscFv format and produced in CHO cells with good yield and purity. Moreover, the bsAb can trigger robust T cell proliferation and activation and induce efficient T cell-mediated MM cell killing in vitro. Using a MM xenograft mouse model, we demonstrate that the bsAb can effectively suppress MM cell growth in vivo. Conclusions: Our results suggest that the BCMA × CD3 bsAb in the FabscFv format can efficiently inhibit MM cell growth and have promising potential to be developed into a therapeutic antibody drug for the treatment of MM.

Excellent removal of knob-into-hole bispecific antibody byproducts and impurities in a single-capture chromatography.

Bispecific antibodies (bsAbs) are therapeutically promising due to their ability to bind to two different antigens. However, the bsAb byproducts and impurities, including mispaired homodimers, half-antibodies, light chain mispairings, antibody fragments and high levels of high molecular weight (HMW) species, all pose unique challenges to their downstream processing. Here, using two knob-into-hole (KiH) constructs of bsAbs as model molecules, we demonstrate the excellent removal of bsAb byproducts and impurities in a single Protein A chromatography under optimized conditions, including hole-hole homodimer mispaired products which are physicochemically very similar to the target bsAbs and still present even with the use of the KiH format, though at reduced levels. The removal occurs through the incorporation of an intermediate low-pH wash step and optimal elution conditions, achieving ~ 60% monomeric purity increase in a single Protein A step, without the introduction of sequence-specific bsAb modifications to specifically induce differential Protein A binding. Our results also suggest that the higher aggregation propensity of bsAbs may cause aggregation during the column process, hence an optimization of the appropriate loading amount, which may be lower than that of monoclonal antibodies (mAbs), is required. With the use of loading at 50% of 10% breakthrough (QB10) at 6-min residence time, we show that an overall high monomer purity of 92.1-93.2% can be achieved with good recovery of 78.4-90.6% within one capture step, which is a significant improvement from a monomer purity of ~ 30% in the cell culture supernatant (CCS). The results presented here would be an insightful guidance to all researchers working on the purification process development to produce bispecific antibodies, especially for knob-into-hole bispecific antibodies.

Effective flow-through polishing strategies for knob-into-hole bispecific antibodies.

Bispecific antibodies (bsAbs), though possessing great therapeutic potential, are extremely challenging to obtain at high purity within a limited number of scalable downstream processing steps. Complementary to Protein A chromatography, polishing strategies play a critical role at removing the remaining high molecular weight (HMW) and low molecular weight (LMW) species, as well as host cell proteins (HCP) in order to achieve a final product of high purity. Here, we demonstrate using two knob-into-hole (KiH) bsAb constructs that two flow-through polishing steps utilising Capto Butyl ImpRes and Capto adhere resins, performed after an optimal Protein A affinity chromatography step can further reduce the HCP by 17- to 35-fold as well as HMW and LMW species with respect to monomer by ~ 4-6% and ~ 1%, respectively, to meet therapeutical requirement at 30-60 mg/mL-resin (R) load. This complete flow-through polishing strategy, guided by Design of Experiments (DoE), eliminates undesirable aggregation problems associated with the higher aggregation propensity of scFv containing bsAbs that may occur in the bind and elute mode, offering an improved ease of overall process operation without additional elution buffer preparation and consumption, thus aligning well with process intensification efforts. Overall, we demonstrate that through the employment of (1) Protein A chromatography step and (2) flow-through polishing steps, a final product containing < 1% HMW species, < 1% LMW species and < 100 ppm HCP can be obtained with an overall process recovery of 56-87%.