Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity

Tustian, Andrew D.; Endicott, Christine; Adams, Benjamin; Mattila, John; Bak, Hanne

doi:10.1080/19420862.2016.1160192

Cited by 87 publications

(82 citation statements)

References 36 publications

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“…This explanation is consistent with the fact that IgG 1 , IgG 2 , and IgG 4 species contain two functional ProA binding sites on the Fc region of the antibody (Moks et al, 1986;Sj€ oquist et al, 1972). In fact, Tustian et al (2016) recently demonstrated that removing one of these Fc binding sites via protein engineering resulted in higher elution pHs, and thus, lower binding strength, from multiple ProA resins due to ligand avidity.…”

Section: Clsm Adsorption Experimentssupporting

confidence: 82%

“…5c and 6c. In fact, Tustian et al (2016) recently demonstrated that removing one of these Fc binding sites via protein engineering resulted in higher elution pHs, and thus, lower binding strength, from multiple ProA resins due to ligand avidity. Since the extent of desorption of the mAb was similar from MabSelect and MabSelect SuRe at 4 h (57 AE 7%), these results suggest that species of equivalent or near equivalent elution pH were also capable of displacing each other on the resin.…”

Section: Clsm Adsorption Experimentsmentioning

confidence: 99%

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Polyclonal and monoclonal IgG binding on protein A resins—Evidence of competitive binding effects

Weinberg

Zhang²,

Crews

et al. 2017

Biotech & Bioengineering

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Protein A (ProA) chromatography is used extensively in the biopharmaceutical industry for the selective capture of both polyclonal and monoclonal antibodies (mAbs). This work provides a comparison of the adsorptive behavior of a highly heterogeneous polyclonal hIgG versus that of a mAb as well as the behavior of their mixtures on representative ProA resins. Both pH gradient elution and frontal loading experiments using human polyclonal IgG (hIgG) reveal a distribution of IgG-ProA binding strengths likely associated with multiple IgG subclasses and the heterogeneity of the variable region. pH gradient analysis of fractions collected along the breakthrough curve demonstrate a clear progression from weaker binding (higher pH eluting) to stronger binding (lower pH eluting) IgG species leaving the column suggesting the possibility of stronger binding species displacing the weaker binding ones. Displacement is directly observed by visualizing the adsorption of fluorescently labeled mAb and hIgG using confocal laser scanning microscopy (CLSM). Here, the displacement of hIgG results in a broad adsorption front compared to the sharp, "shrinking core" behavior typically observed with mAbs. Sequential CLSM adsorption experiments with a mAb and hIgG confirm that stronger or equivalent-binding hIgG species are able to displace and desorb bound mAb molecules. These phenomena are examined using a variety of ProA resins including CaptivA PriMAB, MabSelect, and MabSelect SuRe to understand the effect of different ligand properties on binding strength and competition among different IgG species. The results of these comparisons suggest that the competition kinetics are slower with ligands that have a single-point covalent attachment to the base matrix compared to a multi-point attachment. Biotechnol. Bioeng. 2017;114: 1803-1812. © 2017 Wiley Periodicals, Inc.

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Section: Clsm Adsorption Experimentssupporting

confidence: 82%

Section: Clsm Adsorption Experimentsmentioning

confidence: 99%

Polyclonal and monoclonal IgG binding on protein A resins—Evidence of competitive binding effects

Weinberg

Zhang²,

Crews

et al. 2017

Biotech & Bioengineering

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“…For any 2 antibodies containing a common LC, bsAb purification can be expedited by incorporating mutations that ablate protein A binding into one of the parental HCs. 267 After coexpression of the 2 HCs and a common LC, bsAbs can be purified from the mixture using protein A resin combined with incremental decreases in buffer pH. The species containing 2 mutated HCs flows through the column, while the heterodimer elutes at intermediate pH and the wild-type HC homodimer elutes only at low pH because of strong protein A avidity.…”

Section: Asymmetric Igg-like Frameworkmentioning

confidence: 99%

Considerations for the Design of Antibody-Based Therapeutics

Goulet

Atkins

2020

Journal of Pharmaceutical Sciences

182

154

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Keywords: antibody(s) antibody drug(s) antibody drug conjugate(s) (ADC) physicochemical properties pharmacokinetics monoclonal antibody(s) immunotherapy IgG antibody(s) drug design developability a b s t r a c t Antibody-based proteins have become an important class of biologic therapeutics, due in large part to the stability, specificity, and adaptability of the antibody framework. Indeed, antibodies not only have the inherent ability to bind both antigens and endogenous immune receptors but also have proven extremely amenable to protein engineering. Thus, several derivatives of the monoclonal antibody format, including bispecific antibodies, antibody-drug conjugates, and antibody fragments, have demonstrated efficacy for treating human disease, particularly in the fields of immunology and oncology. Reviewed here are considerations for the design of antibody-based therapeutics, including immunological context, therapeutic mechanisms, and engineering strategies. First, characteristics of antibodies are introduced, with emphasis on structural domains, functionally important receptors, isotypic and allotypic differences, and modifications such as glycosylation. Then, aspects of therapeutic antibody design are discussed, including identification of antigen-specific variable regions, choice of expression system, use of multispecific formats, and design of antibody derivatives based on fragmentation, oligomerization, or conjugation to other functional moieties. Finally, strategies to enhance antibody function through protein engineering are reviewed while highlighting the impact of fundamental biophysical properties on protein developability.

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“…Two different variations of each antibody were cloned to contain either the F405L or K409R mutation to promote cFAE [33]. Furthermore, antibodies containing the K409R mutation also contained the H435R and Y436F mutations to ablate protein A binding in one arm of the heterodimer [34]. Antibodies were cloned into a proprietary AstraZeneca mammalian expression vector [35], and transient expression was carried out using suspension-adapted chinese hamster ovary (CHO) cells [36].…”

Section: Transient Expression Of Parental Antibodiesmentioning

confidence: 99%

“…Here, we describe a method for the rapid generation of monovalent bsAbs directly from culture media by combining a single-matched point mutation in the CH3 domain to promote heterodimerization via controlled Fab-arm exchange (cFAE) [33], and by incorporating the H435R and Y436F mutations in the CH3 domain to ablate protein A binding in one arm of the heterodimer to facilitate bsAb purification [34]. Using this approach, highly pure monovalent bsAbs can be rapidly generated and purified directly from combined culture media using standard protein A purification.…”

Section: Introductionmentioning

confidence: 99%

Fab-Arm Exchange Combined with Selective Protein A Purification Results in a Platform for Rapid Preparation of Monovalent Bispecific Antibodies Directly from Culture Media

Steinhardt

Fleming

et al. 2019

Pharmaceutics

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Bispecific antibody (bsAb) applications have exponentially expanded with the advent of molecular engineering strategies that have addressed many of the initial challenges, including improper light chain pairing, heterodimer purity, aggregation, and pharmacokinetics. However, the lack of high-throughput methods for the generation of monovalent bsAbs has resulted in a bottleneck that has hampered their therapeutic evaluation, as current technologies can be cost-prohibitive and impractical. To address this issue, we incorporated single-matched point mutations in the CH3 domain to recapitulate the physiological process of human IgG4 Fab-arm exchange to generate monovalent bsAbs. Furthermore, we utilized the substitutions H435R and Y436F in the CH3 domain of IgG1, which incorporates residues from human IgG3, thus ablating protein A binding. By exploiting this combination of mutations and optimizing the reduction and reoxidation conditions for Fab arm exchange, highly pure monovalent bsAbs can be rapidly purified directly from combined culture media using standard protein A purification. This methodology, reported herein for the first time, allows for the high-throughput generation of monovalent bsAbs, thus increasing the capacity for evaluating monovalent bsAb iterations for therapeutic potential.

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Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity

Cited by 87 publications

References 36 publications

Polyclonal and monoclonal IgG binding on protein A resins—Evidence of competitive binding effects

Polyclonal and monoclonal IgG binding on protein A resins—Evidence of competitive binding effects

Considerations for the Design of Antibody-Based Therapeutics

Fab-Arm Exchange Combined with Selective Protein A Purification Results in a Platform for Rapid Preparation of Monovalent Bispecific Antibodies Directly from Culture Media

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