Discovery and characterization of hydroxylysine in recombinant monoclonal antibodies

Xie, Qing; Moore, Benjamin; Beardsley, Richard L.

doi:10.1080/19420862.2015.1122148

Cited by 14 publications

(9 citation statements)

References 32 publications

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“…To date, two studies have reported hydroxylation in antibodies. Xie et al reported Lys hydroxylation for an IgG1 antibody, 125 and Tyshchu et al observed Pro hydroxylation for a bispecific antibody. 112 Using consensus motifs can lead to the overestimation of hydroxylation sites.…”

Section: Hydroxylationmentioning

confidence: 99%

“…For example, Xie et al did not report Lys hydroxylation at all of the consensus motifs. 125 Most hydroxylation predictors are not specific to mammalian and human proteins, limiting their utility for therapeutic antibodies. 123 Tyshchuk and colleagues used hydroxylation sites for mammalian proteins to generate sequence logos ( Figure 5(b )) and develop a kNN model: 112 the in silico prediction correlated well with the experimental data for the bispecific antibody.…”

Section: Hydroxylationmentioning

confidence: 99%

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In silico prediction of post-translational modifications in therapeutic antibodies

Vatsa¹

2022

mAbs

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Monoclonal antibodies are susceptible to chemical and enzymatic modifications during manufacturing, storage, and shipping. Deamidation, isomerization, and oxidation can compromise the potency, efficacy, and safety of therapeutic antibodies. Recently, in silico tools have been used to identify liable residues and engineer antibodies with better chemical stability. Computational approaches for predicting deamidation, isomerization, oxidation, glycation, carbonylation, sulfation, and hydroxylation are reviewed here. Although liable motifs have been used to improve the chemical stability of antibodies, the accuracy of in silico predictions can be improved using machine learning and molecular dynamic simulations. In addition, there are opportunities to improve predictions for specific stress conditions, develop in silico prediction of novel modifications in antibodies, and predict the impact of modifications on physical stability and antigen-binding.

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Section: Hydroxylationmentioning

confidence: 99%

Section: Hydroxylationmentioning

confidence: 99%

In silico prediction of post-translational modifications in therapeutic antibodies

Vatsa¹

2022

mAbs

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“…A recent study reported hydroxylation of a lysine residue of an IgG1 catalyzed by lysyl hydroxylase during cell culture . The same study reports identification of hydroxylation of lysine residue at the same location for several additional CHO‐expressed recombinant monoclonal antibodies and hypothesized that this modification could be common to recombinant monoclonal antibodies.…”

Section: Other Modificationsmentioning

confidence: 90%

Impact of cell culture on recombinant monoclonal antibody product heterogeneity

Liu

Nowak

Shao

et al. 2016

Biotechnology Progress

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Recombinant monoclonal antibodies are commonly expressed in mammalian cell culture and purified by several steps of filtration and chromatography. The resulting high purity bulk drug substance still contains product variants differing in properties such as charge and size. Posttranslational modifications and degradations occurring during cell culture are the major sources of heterogeneity in bulk drug substance of recombinant monoclonal antibodies. The focus of the current review is the impact of cell culture conditions on the types and levels of various modifications and degradations of recombinant monoclonal antibodies. Understanding the relationship between cell culture and product variants can help to make consistently safe and efficacious products. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1103-1112, 2016.

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“…Occasionally, unexpected enzyme-catalyzed PTMs, such as hydroxylysine, glutamine-linked glycosylation, and non-consensus Asn-glycosylation, have been reported in constant domains or framework regions of IgGs. [3][4][5] Also, complementarity-determining regions (CDRs), which have inherent variability and solvent exposure, have been identified with unusual enzyme-catalyzed PTMs like O-fucosylation and sulfotyrosine. 6,7 Further uncommon enzyme-catalyzed PTMs (based on the experience and knowledge from IgGs and monoclonal antibodies (mAbs)) have been reported in non-IgG domains and linkers of antibody-fusion proteins.…”

Section: Introductionmentioning

confidence: 99%

Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics

Tyshchuk

Gstöttner

Funk

et al. 2019

mAbs

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Biotherapeutics may contain a multitude of different post-translational modifications (PTMs) that need to be assessed and possibly monitored and controlled to ensure reproducible product quality. During early development of biotherapeutics, unexpected PTMs might be prevented by in silico identification and characterization together with further molecular engineering. Mass determinations of a human IgG1 (mAb1) and a bispecific IgG-ligand fusion protein (BsAbA) demonstrated the presence of unusual PTMs resulting in major +80 Da, and +16/+32 Da chain variants, respectively. For mAb1, analytical cation exchange chromatography demonstrated the presence of an acidic peak accounting for 20%. A + 79.957 Da modification was localized within the light chain complementarity-determining region-2 and identified as a sulfation based on accurate mass, isotopic distribution, and a complete neutral loss reaction upon collision-induced dissociation. Top-down ultrahigh resolution MALDI-ISD FT-ICR MS of modified and unmodified Fabs allowed the allocation of the sulfation to a specific Tyr residue. An aspartate in amino-terminal position-3 relative to the affected Tyr was found to play a key role in determining the sulfation. For BsAbA, a + 15.995 Da modification was observed and localized to three specific Pro residues explaining the +16 Da chain A, and +16 Da and +32 Da chain B variants. The BsAbA modifications were verified as 4-hydroxyproline and not 3-hydroxyproline in a tryptic peptide map via cochromatography with synthetic peptides containing the two isomeric forms. Finally, our approach for an alert system based on in-house in silico predictors is presented. This system is designed to prevent these PTMs by molecular design and engineering during early biotherapeutic development.

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Discovery and characterization of hydroxylysine in recombinant monoclonal antibodies

Cited by 14 publications

References 32 publications

In silico prediction of post-translational modifications in therapeutic antibodies

In silico prediction of post-translational modifications in therapeutic antibodies

Impact of cell culture on recombinant monoclonal antibody product heterogeneity

Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics

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