Alkaline cation-exchange chromatography for the reduction of aggregate and a mis-formed disulfide variant in a bispecific antibody purification process

Hall, Troii; Wilson, Joseph J.; Brownlee, Tammy J.; Swartling, James R.; Langan, Sarah E.; Lambooy, Peter K.

doi:10.1016/j.jchromb.2014.11.002

Cited by 18 publications

(3 citation statements)

References 20 publications

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“…Different strategies were explored previously to enhance aggregate removal in bsAb purification by CEX, among which the use of elevated alkaline pH during elution seemed to be promising, as demonstrated by pH 8.1 for elution (Hall et al 2015 ). However, this method only benefited bsAb molecules with strong alkaline tolerance which limited its application.…”

Section: Discussionmentioning

confidence: 99%

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

Ingavat,

Wang,

Liew

et al. 2023

Bioresour. Bioprocess.

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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. Graphical abstract

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Section: Discussionmentioning

confidence: 99%

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

Ingavat,

Wang,

Liew

et al. 2023

Bioresour. Bioprocess.

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“…Recently, ion-exchange-based methods using membrane technology have become popular, and it is thought that they may soon replace anion-exchange resins. 70 In another study, Hall et al 71 employed cation-exchange chromatography for the resolution of a bispecific antibody and the removal of a misformed mAb-diabody species as well as aggregate products. Furthermore, Müller-Späth et al 6 showed that ionexchange chromatography could be used to fully substitute a costly Protein-A resin for mAb purification.…”

Section: Anion/cation-exchange Chromatographymentioning

confidence: 99%

Technology advancements in antibody purification

Murphy¹,

Devine²,

O’Kennedy³

2016

ANTI

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Abstract:The separation of antibodies from complex mixtures can be achieved using chromatographic or non-chromatographic techniques. The purification of antibodies using chromatography involves the separation of antibodies or antibody-derived molecules present in complex mixtures by passing them through a solid phase (eg, silica resin or beads, monolithic columns, or cellulose membranes) and allowing the antibodies to bind or pass through depending on whether "bind-and-elute" or "flow-through" chromatographic methods are employed. Chromatographic methodologies can incorporate different separation techniques, such as affinity-tag binding, ion-exchange, size-exclusion chromatography, or immunoaffinity chromatography. However, in a concerted effort to become less reliant on chromatography-based separations owing to the high cost of large-scale production, non-chromatographic-based techniques such as precipitation, flocculation, crystallization, filtration, and aqueous two-phase partitioning are now becoming more popular. This review details current chromatographic and non-chromatographic methodo logies used for the purification of antibodies and expands on technological advancements and practical uses that have recently been reported.

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“…Besides size-based techniques which provide separation based on the hydrodynamic radius, many of the currently reported protocols are performed in the bind and elute mode, where the target molecule bound on the stationary phase is eluted via alterations often in the pH or salt concentration (Chen and Zhang 2021 ; Li et al 2020 ). Charge-based purification is frequently employed in the form of ion exchange chromatography (Andrade et al 2019 ; Jakobsen 2011 ; Vallera and Miller 2017 ; Guo et al 2020 ; Allan et al 2014 ; Brischwein et al 2006 ; Kimerer et al 2019 ; Sharkey et al 2017 ; Igawa and Tsunoda 2009 ; Sampei et al 2013 ; Hall et al 2015 ), where bsAbs are commonly loaded in the absence of salt at a recommended pH of 1 to 3 units away from the isoelectric point (pI) of the target molecule followed by elution with buffer with high salt concentrations. Alternatively, the target molecule may be eluted with pH close to the target pI or with a combination of salt and pH effects.…”

Section: Introductionmentioning

confidence: 99%

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

Chen

Hoi

Mahfut

et al. 2022

Bioresour. Bioprocess.

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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

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Alkaline cation-exchange chromatography for the reduction of aggregate and a mis-formed disulfide variant in a bispecific antibody purification process

Cited by 18 publications

References 20 publications

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

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

Technology advancements in antibody purification

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

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