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

Abstract

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%. Graphical Abstract

Harnessing Ceramic Hydroxyapatite as an Effective Polishing Strategy to Remove Product- and Process-related Impurities in Bispecific Antibody Purification

“…Considering the differences between mAb and bsAb, traditional puri cation tools for mAbs are less likely to provide the same level of e ciency when it comes to bsAbs. In addition, "chromatographyinduced aggregation" has been observed during both Protein A and cation exchange chromatography (CEX) for bsAb puri cation as reported by Serene Chen et al, and Lucus K. Kimerer et al (Chen et al, 2022a(Chen et al, , 2022bKimerer et al, 2019). This "chromatography-induced aggregation" is more pronounced when bsAbs are subjected to high loading (Chen et al, 2022a, b).…”

“…In general, bsAbs are highly aggregation-prone proteins, especially for the scFv-containing molecules (Chen et al, 2022a(Chen et al, , 2022bLi et al, 2016). Chromatography-induced aggregation for both Molecule A and B have been previously reported, when performing a bind-elute mode for both protein A and CEX chromatography (Chen et al, 2022a, b).…”

“…In addition, "chromatographyinduced aggregation" has been observed during both Protein A and cation exchange chromatography (CEX) for bsAb puri cation as reported by Serene Chen et al, and Lucus K. Kimerer et al (Chen et al, 2022a(Chen et al, , 2022bKimerer et al, 2019). This "chromatography-induced aggregation" is more pronounced when bsAbs are subjected to high loading (Chen et al, 2022a, b). This could be a crucial bottleneck for bsAb industrial production as lower loading leads to lower productivity.…”

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

“…Considering the differences between mAb and bsAb, traditional purification tools for mAbs are less likely to provide the same level of efficiency when it comes to bsAbs. In addition, “chromatography-induced aggregation” has been observed during both Protein A and cation exchange chromatography (CEX) for bsAb purification as reported by Serene Chen et al and Lucus K. Kimerer et al (Chen et al 2022a , 2022b ; Kimerer et al 2019 ). This “chromatography-induced aggregation” is more pronounced when bsAbs are subjected to high loading (Chen et al 2022a , 2022b ).…”

“…In addition, “chromatography-induced aggregation” has been observed during both Protein A and cation exchange chromatography (CEX) for bsAb purification as reported by Serene Chen et al and Lucus K. Kimerer et al (Chen et al 2022a , 2022b ; Kimerer et al 2019 ). This “chromatography-induced aggregation” is more pronounced when bsAbs are subjected to high loading (Chen et al 2022a , 2022b ). This could be a crucial bottleneck for bsAb industrial production as lower loading leads to lower productivity.…”