Assessing analytical methods to monitor isoAsp formation in monoclonal antibodies

Eakin, Catherine M.; Miller, Amanda; Kerr, Jennifer; Kung, James Kai-sing; Wallace, Alison

doi:10.3389/fphar.2014.00087

Cited by 29 publications

(35 citation statements)

References 36 publications

(46 reference statements)

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“… 21 Protein-based bioconjugates represent an increasingly popular choice for pharmaceuticals, 22 , 23 accentuating the need for better understanding of effective protein lifetime and degradation byproducts. For example, perturbations to complementarity-determining regions (CDRs) of antibodies can drastically reduce antigen binding efficacy, 24 as demonstrated by deactivation of monoclonal immunoglobulin γ2 following a single isomerization. 25 Furthermore, deamidation is facilitated by the solvent exposed and flexible nature of CDRs.…”

mentioning

confidence: 99%

Sequence and Solution Effects on the Prevalence of d-Isomers Produced by Deamidation

2017

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Deamidation of asparagine is a spontaneous and irreversible post-translational modification associated with a growing list of human diseases. While pervasive, deamidation is often overlooked because it represents a relatively minor chemical change. Structural and functional characterization of this modification is complicated because deamidation of asparagine yields four isomeric forms of Asp. Herein, radical directed dissociation (RDD), in conjunction with mass spectrometry, is used to identify and quantify all four isomers in a series of model peptides that were subjected to various deamidation conditions. Although primary sequence significantly influences the rate of deamidation, it has little impact on the relative proportions of the product isomers. Furthermore, the addition of ammonia can be used to increase the rate of deamidation without significantly perturbing isomer populations. Conversely, external factors such as buffer conditions and temperature alter product distributions but exhibit less dramatic effects on the deamidation rate. Strikingly, the common laboratory and biologically significant bicarbonate buffer is found to strongly promote racemization, yielding increased amounts of d-Asp and d-isoAsp. These outcomes following deamidation have broad implications in human aging and should be considered during the development of protein-based therapeutics.

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confidence: 99%

Sequence and Solution Effects on the Prevalence of d-Isomers Produced by Deamidation

2017

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“…Previous reports have shown that an isoAsp-containing peptide elutes earlier than the corresponding Asp-containing peptide when separated by RP chromatography as a result of changes to the local protein structure. 3,17,25 Intact mass analysis of these 2 elution peaks revealed that the corresponding peptides had the same mass (Fig. 2).…”

Section: Characterization Of Asp Isomerization In Mab1mentioning

confidence: 93%

“…2,12,13 Accelerated stress conditions coupled with various analytical methods have been described to generate, isolate, and characterize Asp isoforms to understand developability risks. 9,10,[14][15][16][17][18] The most common approach for identifying and quantifying Asp isomerization in a mAb involves enzymatic digestion of accelerated stress samples, separation of peptides by reversed phase (RP) liquid chromatography, and subsequent characterization by mass spectrometry.…”

Section: Introductionmentioning

confidence: 99%

N+1 Engineering of an Aspartate Isomerization Hotspot in the Complementarity-Determining Region of a Monoclonal Antibody

Patel

Bauer

Davies

et al. 2016

Journal of Pharmaceutical Sciences

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Aspartate (Asp) isomerization is a common degradation pathway and a potential critical quality attribute that needs to be well characterized during the optimization and development of therapeutic antibodies. A putative Asp-serine (Ser) isomerization motif was identified in the complementarity-determining region of a humanized monoclonal antibody and shown to be a developability risk using accelerated stability analyses. To address this issue, we explored different antibody engineering strategies. Direct engineering of the Asp residue resulted in a greater than 5× loss of antigen-binding affinity and bioactivity, indicating a critical role for this residue. In contrast, rational engineering of the Ser residue at the n+1 position had a negligible impact on antigen binding affinity and bioactivity compared with the parent molecule. Furthermore, the n+1 engineering strategy effectively eliminated Asp isomerization as determined by accelerated stability analysis. This outcome affirms that the rate of Asp isomerization is strongly dependent on the identity of the n+1 residue. This report highlights a systematic antibody engineering strategy for mitigating an Asp isomerization developability risk during lead optimization.

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“…To achieve better resolution and more accurate quantitation, molecules can be broken into their subunits before separation, such as cleaving antibodies into (Fab) 2 and Fc. 36,37 This subunit approach nevertheless is molecule-specific and often requires extensive method development and optimization to achieve desired outcome, adding significant burden to analytical development. An alternative approach to these methods is tryptic peptide mapping (TPM) by LC coupled with MS that can be extremely powerful in probing protein PTMs as it provides comprehensive information on deamidation, oxidation, isomerization, glycan profiles, etc.…”

Section: Introductionmentioning

confidence: 99%

Structure-Function Assessment and High-Throughput Quantification of Site-Specific Aspartate Isomerization in Monoclonal Antibody Using a Novel Analytical Tool Kit

Zhou

Cao

Bautista

et al. 2020

Journal of Pharmaceutical Sciences

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Isomerization of surface-exposed aspartic acid (Asp) in the complementarity-determining regions of therapeutic proteins could potentially impact their target binding affinity because of the sensitive location, and often requires complex analytical tactics to understand its effect on structure-function and stability. Inaccurate quantitation of Asp-isomerized variants, especially the succinimide intermediate, presents major challenge in understanding Asp degradation kinetics, its stability, and consequently establishing a robust control strategy. As a practical solution to this problem, a comprehensive analytical tool kit has been developed, which provides a solution to fully characterize and accurately quantify the Asp-related product variants. The toolkit offers a combination of 2 steps, an ion-exchange chromatography method to separate and enrich the isomerized variants in the folded structure for structurefunction evaluation and a novel focused peptide mapping method to quantify the individual complementarity-determining region isomerization components including the unmodified Asp, succinimide, and isoaspartate. This novel procedure allowed an accurate quantification of each Asp-related variant and a comprehensive assessment of the functional impact of Asp isomerization, which ultimately helped to establish an appropriate control strategy for this critical quality attribute.

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Assessing analytical methods to monitor isoAsp formation in monoclonal antibodies

Cited by 29 publications

References 36 publications

Sequence and Solution Effects on the Prevalence of d-Isomers Produced by Deamidation

Sequence and Solution Effects on the Prevalence of d-Isomers Produced by Deamidation

N+1 Engineering of an Aspartate Isomerization Hotspot in the Complementarity-Determining Region of a Monoclonal Antibody

Structure-Function Assessment and High-Throughput Quantification of Site-Specific Aspartate Isomerization in Monoclonal Antibody Using a Novel Analytical Tool Kit

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