Elucidation of Degradants in Acidic Peak of Cation Exchange Chromatography in an IgG1 Monoclonal Antibody Formed on Long-Term Storage in a Liquid Formulation

Gandhi, Sejal P.; Ren, Da; Xiao, Gang; Bondarenko, Pavel V.; Sloey, Christopher; Ricci, Margaret Speed; Krishnan, Sampathkumar

doi:10.1007/s11095-011-0536-0

Cited by 55 publications

(30 citation statements)

References 50 publications

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“…The dilution of the pharmaceutical product, which contains stabilizing components (neutral detergent) caused a slow formation of acidic variants, while the amount of the basic variants did not change. Mainly the acidic variants in the drug product could have impact on biological activity, so the fast and reliable determination of the amounts of these variants is required [17]. The electrophoretic patterns presented in (Figure 1 c) are in good agreement with the electropherogram obtained in neutral coated capillary [16].…”

Section: Cze Separation Of Rituximab In Uncoated Capillarysupporting

confidence: 62%

Analysis of Rituximab, A Therapeutic Monoclonal Antibody by Capillary Zone Electrophoresis

G¹

2014

J Chromatogr Sep Tech

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This paper focuses on the applicability of capillary zone electrophoresis (CZE) using uncoated fused silica capillaries for the determination of heterogeneity of rituximab (MabThera, Roche) and for the study of its solution and thermal stability. The best resolution for the charge variants of the main component was obtained with a buffer electrolyte containing 800 mM 6-amino caproic acid, 2 mM triethylene tetramine and 0.05% hydroxypropyl methylcellulose at pH = 5.2. It was found that the pH and the components of the buffer used for the electrophoretic separation and for the dilution of the sample prior to the analysis are important to the stability of the rituximab. We demonstrated the rituximab is stable in the pharmaceutical product MabThera due to the stabilizing additives, but the dilution of the MabThera caused a slow formation of acidic variants, while the amount of the basic variants did not change. After incubation of the diluted rituximab at higher temperature several charge variants could be determined by CZE.

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Section: Cze Separation Of Rituximab In Uncoated Capillarysupporting

confidence: 62%

Analysis of Rituximab, A Therapeutic Monoclonal Antibody by Capillary Zone Electrophoresis

G¹

2014

J Chromatogr Sep Tech

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“…27, and no impact of deamidation on FcRn binding at pH 6 was found. Here, no impact of deamidation on FcγRI (pH 7.4) and FcRn (both pH 6 and pH 7.4) was measured.…”

Section: Discussionmentioning

confidence: 88%

Rapid screening of IgG quality attributes – effects on Fc receptor binding

Geuijen¹,

Oppers-Tiemissen

Egging

et al. 2017

FEBS Open Bio

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The interactions of therapeutic antibodies with fragment crystallizable γ (Fcγ) receptors and neonatal Fc receptors (FcRn) are measured in vitro as indicators of antibody functional performance. Antibodies are anchored to immune cells through the Fc tail, and these interactions are important for the efficacy and safety of therapeutic antibodies. High‐throughput binding studies on each of the human Fcγ receptor classes (FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb) as well as FcRn have been developed and performed with human IgG after stress‐induced modifications to identify potential impact in vivo. Interestingly, we found that asparagine deamidation (D‐N) reduced the binding of IgG to the low‐affinity Fcγ receptors (FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb), while FcγRI and FcRn binding was not impacted. Deglycosylation completely inhibited binding to all Fcγ receptors, but showed no impact on binding to FcRn. On the other hand, afucosylation only impacted binding to FcγRIIIa and FcγRIIIb. Methionine oxidation at levels below 7%, multiple freeze/thaw cycles and short‐term thermal/shake stress did not influence binding to any of the Fc receptors. The presence of high molecular weight species, or aggregates, disturbed measurements in these binding assays; up to 5% of aggregates in IgG samples changed the binding and kinetics to each of the Fc receptors. Overall, the screening assays described in this manuscript prove that rapid and multiplexed binding assays may be a valuable tool for lead optimization, process development, in‐process controls, and biosimilarity assessment of IgGs during development and manufacturing of therapeutic IgGs.

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“…This technique was classically used in studies of multiple myeloma (4) and is currently used for analyzing the degradation of monoclonal antibodies (14,24,61). IEF has also been used in the study of the humoral response to a number of pathogens (10, 11, 15, 17, 20-22, 30, 37, 39, 45, 46, 50, 51, 55).…”

Section: Discussionmentioning

confidence: 99%

Signature Biochemical Properties of Broadly Cross-Reactive HIV-1 Neutralizing Antibodies in Human Plasma

Sajadi

Lewis

Seaman

et al. 2012

J Virol

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The common properties of broadly cross-reactive HIV-1 neutralization antibodies found in certain HIV-1-infected individuals holds significant value for understanding natural and vaccine-mediated anti-HIV immunity. Recent efforts have addressed this question by deriving neutralizing monoclonal anti-envelope antibodies from memory B cell pools of selected subjects. However, it has been more difficult to identify whether broadly neutralizing antibodies circulating in plasma possess shared characteristics among individuals. To address this question, we used affinity chromatography and isoelectric focusing to fractionate plasma immunoglobulin from 10 HIV-1-infected subjects (5 subjects with broad HIV-1 neutralizing activity and 5 controls). We find that plasma neutralizing activity typically partitions into at least two subsets of antibodies. Antibodies with restricted neutralization breadth have relatively neutral isoelectric points and preferentially bind to envelope monomers and trimers versus core antigens from which variable loops and other domains have been deleted. In comparison, broadly neutralizing antibodies account for a minor fraction of the total anti-envelope response. They are consistently distinguished by more basic isoelectric points and specificity for epitopes shared by monomeric gp120, gp120 core, or CD4-induced structures. Such biochemical properties might be exploited to reliably predict or produce broad anti-HIV immunity.

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Elucidation of Degradants in Acidic Peak of Cation Exchange Chromatography in an IgG1 Monoclonal Antibody Formed on Long-Term Storage in a Liquid Formulation

Abstract: The observed CEX pre-peak increase was caused by multiple degradations, especially deamidation and clipping. This elucidation of degradants in CEX peaks may apply to other therapeutic IgG1 monoclonal antibodies.

Cited by 55 publications

References 50 publications

Analysis of Rituximab, A Therapeutic Monoclonal Antibody by Capillary Zone Electrophoresis

Analysis of Rituximab, A Therapeutic Monoclonal Antibody by Capillary Zone Electrophoresis

Rapid screening of IgG quality attributes – effects on Fc receptor binding

Signature Biochemical Properties of Broadly Cross-Reactive HIV-1 Neutralizing Antibodies in Human Plasma

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