Investigation of N-terminal glutamate cyclization of recombinant monoclonal antibody in formulation development

Liu, Yu; Vizel, A. O.; Huff, Mary Beth; Young, M.W.; Remmele, Richard L.; He, Bing

doi:10.1016/j.jpba.2006.05.008

Cited by 73 publications

(72 citation statements)

References 18 publications

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“…Literature reports also have documented water loss in N-terminal E residues in proteins, including antibodies. [63][64][65] Thus, the MS data supports the hypothesis that a 5-membered ring can form to interfere with N-terminal carbodiimide labeling. Although a Dresidue in a corresponding position cannot form such a ring, some suppression is still observed, suggesting that both ring formation and the N-terminal positive charge can suppress reactivity, but that the latter effect is less than the former.…”

Section: Results From D-peptidesupporting

confidence: 73%

Characterizing monoclonal antibody structure by carbodiimide/GEE footprinting

Kaur

Tomechko

Kiselar

et al. 2014

mAbs

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Amino acid-specific covalent labeling is well suited to probe protein structure and macromolecular interactions, especially for macromolecules and their complexes that are difficult to examine by alternative means, due to size, complexity, or instability. Here we present a detailed account of carbodiimide-based covalent labeling (with GEE tagging) applied to a glycosylated monoclonal antibody therapeutic, which represents an important class of biologic drugs. Characterization of such proteins and their antigen complexes is essential to development of new biologic-based medicines. In this study, the experiments were optimized to preserve the structural integrity of the protein, and experimental conditions were varied and replicated to establish the reproducibility and precision of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include D, E, and the C-terminus, against the experimental surface accessibility data in order to understand the accuracy of the approach in providing an unbiased assessment of structure. Data from the protein were also compared to reactivity measures of several model peptides to explain sequence or structure-based variations in reactivity. The results highlight several advantages of this approach. These include: the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling (indicating that the label does not significantly perturb the structure of the protein), the high reproducibility of replicate experiments (<2 % variation in modification extent), the similar reactivity of the 3 target probe residues (as suggested by analysis of model peptides), and the overall positive and significant correlation of reactivity and solvent accessible surface area (the latter values predicted by the homology modeling). Attenuation of reactivity, in otherwise solvent accessible probes, is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. The results are also compared with data from hydroxyl radical-mediated oxidative footprinting on the same protein, showing that complementary information is gained from the 2 approaches, although the number of target residues in carbodiimide/GEE labeling is fewer. Overall, this approach is an accurate and precise method for assessing protein structure of biologic drugs. IntroductionMethods to describe structures of proteins and their higher order complexes continue to evolve and various advances have increased speed, efficiency, and resolution of structure determination.1 Most proteins behave according to the functiondictated-by-structure principle, and their proper conformation is central to their role in processes such as enzyme catalysis, cell signaling, or ligand binding. [2][3][4] Protein drugs (biologics) are one of the most important and fastest growing classes of drugs in the commercial marketplace today. The function and efficacy...

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Section: Results From D-peptidesupporting

confidence: 73%

Characterizing monoclonal antibody structure by carbodiimide/GEE footprinting

Kaur

Tomechko

Kiselar

et al. 2014

mAbs

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“…With respect to the primary structure of HBSP, however, it is well known that N-terminal glutamine residues can undergo a spontaneous, irreversible cyclization (particularly at room temperature under acidic conditions) into pyroglutamate (32). In confirmation of this fact, amino acid analysis of production batches of HBSP revealed that Ϸ90% of the product possessed a free N-terminal glutamine, whereas the remainder was cyclized.…”

Section: Resultsmentioning

confidence: 99%

Nonerythropoietic, tissue-protective peptides derived from the tertiary structure of erythropoietin

Brines¹,

Patel

Villa

et al. 2008

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

280

283

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Erythropoietin (EPO), a member of the type 1 cytokine superfamily, plays a critical hormonal role regulating erythrocyte production as well as a paracrine/autocrine role in which locally produced EPO protects a wide variety of tissues from diverse injuries. Significantly, these functions are mediated by distinct receptors: hematopoiesis via the EPO receptor homodimer and tissue protection via a heterocomplex composed of the EPO receptor and CD131, the ␤ common receptor. In the present work, we have delimited tissueprotective domains within EPO to short peptide sequences. We demonstrate that helix B (amino acid residues 58 -82) of EPO, which faces the aqueous medium when EPO is bound to the receptor homodimer, is both neuroprotective in vitro and tissue protective in vivo in a variety of models, including ischemic stroke, diabetes-induced retinal edema, and peripheral nerve trauma. Remarkably, an 11-aa peptide composed of adjacent amino acids forming the aqueous face of helix B is also tissue protective, as confirmed by its therapeutic benefit in models of ischemic stroke and renal ischemia-reperfusion. Further, this peptide simulating the aqueous surface of helix B also exhibits EPO's trophic effects by accelerating wound healing and augmenting cognitive function in rodents. As anticipated, neither helix B nor the 11-aa peptide is erythropoietic in vitro or in vivo. Thus, the tissue-protective activities of EPO are mimicked by small, nonerythropoietic peptides that simulate a portion of EPO's three-dimensional structure.cognition ͉ cytoprotection ͉ excitotoxicity ͉ ischemia-reperfusion injury ͉ wound healing

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“…While the non-enzymatical cyclization reaction of Gln to PyroGlu seems to occur predominantly during protein production in cell culture, [63] the chemical conversion of Glu to PyroGlu happens later during processing and storage of the product and seems to be heavily dependent on buffer conditions. [64,65] Additionally, both reactions can also occur in an enzymatically driven fashion, catalyzed by the glutaminyl cyclase. [66] Non-enzymatic conversion of glutamate to pyroglutamate has also been observed on antibodies in vivo, without affecting the pharmacological properties of the molecules, such as its safety, pharmacodynamics, and pharmacokinetics.…”

Section: N-terminal Pyroglutamate Formationmentioning

confidence: 99%

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

Beyer

Schuster

Jungbauer

et al. 2017

Biotechnology Journal

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Antibodies are typical examples of biopharmaceuticals which are composed of numerous, almost infinite numbers of potential molecular entities called variants or isoforms, which constitute the microheterogeneity of these molecules. These variants are generated during biosynthesis by so‐called posttranslational modification, during purification or upon storage. The variants differ in biological properties such as pharmacodynamic properties, for example, Antibody Dependent Cellular Cytotoxicity, complement activation, and pharmacokinetic properties, for example, serum half‐life and safety. Recent progress in analytical technologies such as various modes of liquid chromatography and mass spectrometry has helped to elucidate the structure of a lot of these variants and their biological properties. In this review the most important modifications (glycosylation, terminal modifications, amino acid side chain modifications, glycation, disulfide bond variants and aggregation) are reviewed and an attempt is made to give an overview on the biological properties, for which the reports are often contradictory. Even though there is a deep understanding of cellular and molecular mechanism of antibody modification and their consequences, the clinical proof of the effects observed in vitro and in vivo is still not fully rendered. For some modifications such as core‐fucosylation of the N‐glycan and aggregation the effects are clear and should be monitored, but with others such as C‐terminal lysine clipping the reports are contradictory. As a consequence it seems too early to tell if any modification can be safely ignored.

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Investigation of N-terminal glutamate cyclization of recombinant monoclonal antibody in formulation development

Cited by 73 publications

References 18 publications

Characterizing monoclonal antibody structure by carbodiimide/GEE footprinting

Characterizing monoclonal antibody structure by carbodiimide/GEE footprinting

Nonerythropoietic, tissue-protective peptides derived from the tertiary structure of erythropoietin

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

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