HIC resolution of an IgG1 with an oxidized Trp in a complementarity determining region

Boyd, Daniel; Kaschak, Timothy; Yan, Boxu

doi:10.1016/j.jchromb.2011.03.006

Cited by 69 publications

(61 citation statements)

References 29 publications

(34 reference statements)

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“…Forced oxidation studies, isolation and characterization by LC-MS peptide mapping of the species eluting under each peaks were performed. Results show prepeak 2 to contain mAb species oxidized at about 50% of the heavy chain tryptophan 47 residues; a figure consistent with the oxidation of one site per mAb 6 . Oxidation of two of the strictly conserved Fc methionines is also observed in both pre-peaks ( Fig.…”

Section: Oxidation Of Tryptophan In Cdrs Of a Monoclonal Antibodysupporting

confidence: 74%

“…However, low throughput and complex sample preparation may limit the use of this method during process development. In a recent publication, HIC-HPLC was shown to offer ease of use and reasonable selectivity to monitor the oxidation of tryptophan residues located in the CDR of a recombinant mAb 6 . The elution profile presents two well-resolved pre-peaks eluting before the main.…”

Section: Oxidation Of Tryptophan In Cdrs Of a Monoclonal Antibodymentioning

confidence: 99%

“…Oxidation of two of the strictly conserved Fc methionines is also observed in both pre-peaks ( Fig. 2 ) 6 . The method uses two 100 mm Dionex Propac HIC-10 columns in series to improve recovery and resolution 6 .…”

Section: Oxidation Of Tryptophan In Cdrs Of a Monoclonal Antibodymentioning

confidence: 99%

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Separation of mAbs molecular variants by analytical hydrophobic interaction chromatography HPLC: overview and applications

Haverick

Mengisen

Shameem

et al. 2014

mAbs

113

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Hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) is a powerful analytical method used for the separation of molecular variants of therapeutic proteins. The method has been employed for monitoring various post-translational modifications, including proteolytic fragments and domain misfolding in etanercept (Enbrel®); tryptophan oxidation, aspartic acid isomerization, the formation of cyclic imide, and α amidated carboxy terminus in recombinant therapeutic monoclonal antibodies; and carboxy terminal heterogeneity and serine fucosylation in Fc and Fab fragments. HIC-HPLC is also a powerful analytical technique for the analysis of antibody-drug conjugates. Most current analytical columns, methods, and applications are described, and critical method parameters and suitability for operation in regulated environment are discussed, in this review.

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Section: Oxidation Of Tryptophan In Cdrs Of a Monoclonal Antibodysupporting

confidence: 74%

Section: Oxidation Of Tryptophan In Cdrs Of a Monoclonal Antibodymentioning

confidence: 99%

See 1 more Smart Citation

Separation of mAbs molecular variants by analytical hydrophobic interaction chromatography HPLC: overview and applications

Haverick

Mengisen

Shameem

et al. 2014

mAbs

113

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show abstract

“…Although it is known that amino acid oxidation can occur during sample preparation, it is tempting to speculate that the oxidative modification of certain circulating IgGs may have resulted from in vivo posttranslational modifications rather than processing artifacts, because plasma cells experience high levels of oxidative stress (34), and the t 1/2 of circulating Igs in serum is >1 wk, resulting in extensive exposure of antibodies to oxidizing conditions. L-Methionine oxidation has also been observed repeatedly in recombinantly expressed therapeutic antibodies from CHO cells (35,36). Additional studies will be needed to determine the extent of L-Methionine oxidation during sample processing and the frequency of in vivo modification.…”

Section: Discussionmentioning

confidence: 99%

Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response

Wine

Boutz

Lavinder

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

128

156

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We have developed and validated a methodology for determining the antibody composition of the polyclonal serum response after immunization. Pepsin-digested serum IgGs were subjected to standard antigen-affinity chromatography, and resulting elution, wash, and flow-through fractions were analyzed by bottom-up, liquid chromatography-high-resolution tandem mass spectrometry. Identification of individual monoclonal antibodies required the generation of a database of IgG variable gene (V-gene) sequences constructed by NextGen sequencing of mature B cells. Antibody V-gene sequences are characterized by short complementarity determining regions (CDRs) of high diversity adjacent to framework regions shared across thousands of IgGs, greatly complicating the identification of antigen-specific IgGs from proteomically observed peptides. By mapping peptides marking unique V H CDRH3 sequences, we identified a set of V-genes heavily enriched in the affinity chromatography elution, constituting the serum polyclonal response. After booster immunization in a rabbit, we find that the antigenspecific serum immune response is oligoclonal, comprising antibodies encoding 34 different CDRH3s that group into 30 distinct antibody V H clonotypes. Of these 34 CDRH3s, 12 account for ∼60% of the antigen-specific CDRH3 peptide mass spectral counts. For comparison, antibodies with 18 different CDRH3s (12 clonotypes) were represented in the antigen-specific IgG fraction from an unimmunized rabbit that fortuitously displayed a moderate titer for BSA. Proteomically identified antibodies were synthesized and shown to display subnanomolar affinities. The ability to deconvolute the polyclonal serum response is likely to be of key importance for analyzing antibody responses after vaccination and for more completely understanding adaptive immune responses in health and disease.antibody proteomics | antibody repertoire | serum immunoprofiling | B-cell response | humoral response T he first Nobel Prize in Medicine was awarded to Emil von Behring, who in collaboration with Kitasato Shibasaburo and Paul Ehrlich discovered serum antitoxins (1, 2). Remarkably, after more than 100 y of intense research in immunology, little is known about the clonality, relative concentrations, and binding properties of the monoclonal antibodies that constitute the antigen-specific Ig pool in serum.At steady state, circulating antibodies are produced by terminally differentiated B lymphocytes (plasma cells) within the bone marrow, and thus cannot be accessed in living individuals (3). Although recent single B-cell cloning methods (4, 5) have led to the identification of peripheral antigen-specific B memory and/or antibody-secreting cells (plasmablasts), it is generally unknown whether the Igs encoded by peripheral blood B cells correspond to the antibodies present in circulation and especially whether they are present at physiologically relevant levels (i.e., at serum concentrations above K D corresponding to >1 μg/mL for an average affinity of individual antibodies of 5 nM)...

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“…31,32 Analytical HIC is used for understanding stability in different formulations because highly hydrophobic proteins have a higher likelihood to self-associate and form aggregates. In addition, there are many examples of the use of HIC for analytical purposes to resolve PTMs such as N-terminal glutamine cyclization, oxidation of tryptophan and methionine residues, and deamidation.…”

Section: Introductionmentioning

confidence: 99%

Comparison of the in vitro and in vivo stability of a succinimide intermediate observed on a therapeutic IgG1 molecule

Ouellette

Chumsae²,

Clabbers³

et al. 2013

mAbs

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HIC resolution of an IgG1 with an oxidized Trp in a complementarity determining region

Cited by 69 publications

References 29 publications

Separation of mAbs molecular variants by analytical hydrophobic interaction chromatography HPLC: overview and applications

Separation of mAbs molecular variants by analytical hydrophobic interaction chromatography HPLC: overview and applications

Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response

Comparison of the in vitro and in vivo stability of a succinimide intermediate observed on a therapeutic IgG1 molecule

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