Identification of multiple sources of charge heterogeneity in a recombinant antibody

Harris, Reed J.; Kabakoff, Bruce; Macchi, Frank; Shen, Felicity J.; Kwong, May Y.; Andya, James D.; Shire, Steven J.; Bjork, Nancy; Tótpál, Klára; Chen, Anthony B.

doi:10.1016/s0378-4347(00)00548-x

Cited by 481 publications

(529 citation statements)

References 30 publications

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“…A commercial IgG1 antibody Herceptin (calculated MW ϭ 148,058 Da, Genentech, San Francisco, CA) [29]°and°a chimeric IgG2 antibody produced at Amgen (calculated MW ϭ 147,353 Da, Amgen Inc., Thousand Oaks, CA) were used in this study. The IgG2 antibody was subjected to an accelerated stability study under elevated temperatures, which caused degradations of the molecule.…”

Section: Methodsmentioning

confidence: 99%

Trap for MAbs: Characterization of intact monoclonal antibodies using reversed-phase HPLC on-line with ion-trap mass spectrometry

Bondarenko

2005

J. Am. Soc. Mass Spectrom.

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For the first time, the characterization of intact 150-kDa monoclonal antibodies (MAbs) using a commercially available three-dimensional ion-trap mass spectrometer (IT-MS) is reported. The IT-MS analysis was performed on-line with reversed-phase high performance liquid chromatography (RP-HPLC) on a POROS column using a nontraditional solvent system of acetonitrile, isopropanol, ethanol, and water in formic acid. The operating parameters of the IT-MS were optimized by extending the mass range to m/z 4000 and elevating the tube lens offset voltage value to around Ϫ100 V. Mass accuracy better than 300 ppm (Ϯ40 Da) has been routinely achieved for these macromolecules. Multiple peaks 162 Da apart due to the hexose variants of the monoclonal IgG antibodies were partially resolved in mass spectra. Several commercial and chimeric antibodies have been investigated in this study. (J Am Soc Mass Spectrom 2005, 16, 307-311)

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Section: Methodsmentioning

confidence: 99%

Trap for MAbs: Characterization of intact monoclonal antibodies using reversed-phase HPLC on-line with ion-trap mass spectrometry

Bondarenko

2005

J. Am. Soc. Mass Spectrom.

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“…A label may directly perturb the function of a protein, but the reaction conditions used to introduce the label may inadvertently promote some undesirable change such as oxidation, deamidation, side-chain isomerization, or aggregation (11). The basic absence of gross changes in pharmacokinetics or molecular weight are not always sufficient characterization of labeled proteins, and binding or other functional assays may be needed to assess the integrity of an imaging probe.…”

Section: Topics Labeling: First Do No Harmmentioning

confidence: 99%

Tissue Distribution Studies of Protein Therapeutics Using Molecular Probes: Molecular Imaging

Williams¹

2012

AAPS J

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Abstract. Molecular imaging techniques for protein therapeutics rely on reporter labels, especially radionuclides or sometimes near-infrared fluorescent moieties, which must be introduced with minimal perturbation of the protein's function in vivo and are detected non-invasively during whole-body imaging. PET is the most sensitive whole-body imaging technique available, making it possible to perform biodistribution studies in humans with as little as 1 mg of injected antibody carrying 1 mCi (37 MBq) of zirconium-89 radiolabel. Different labeling chemistries facilitate a variety of optical and radionuclide methods that offer complementary information from microscopy and autoradiography and offer some trade-offs in whole-body imaging between cost and logistic difficulty and image quality and sensitivity (how much protein needs to be injected). Interpretation of tissue uptake requires consideration of label that has been catabolized and possibly residualized. Image contrast depends as much on background signal as it does on tissue uptake, and so the choice of injected dose and scan timing guides the selection of a suitable label and helps to optimize image quality. Although only recently developed, zirconium-89 PET techniques allow for the most quantitative tomographic imaging at millimeter resolution in small animals and they translate very well into clinical use as exemplified by studies of radiolabeled antibodies, including trastuzumab in breast cancer patients, in The Netherlands.

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“…Therapeutic mAbs are multi-domain and multi-chain glycosylated proteins, which exhibit significant heterogeneity in structure and stability. [7][8][9][10] Specifically, the Fab and Fc regions of mAbs bear different functionality or surface properties and are expected to respond differently to various stress conditions. Therefore, these regions may contribute differently to protein stability, aggregation, and phase separation.…”

Section: Introductionmentioning

confidence: 99%

The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

et al. 2010

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This study introduces a novel analytical approach for studying aggregation and phase separation of monoclonal antibodies (mAbs). The approach is based on using analytical scale cation-exchange chromatography (CEX) for measuring the loss of soluble monomer in the case of individual and mixed protein solutions. Native CEX outperforms traditional size-exclusion chromatography in separating complex protein mixtures, offering an easy way to assess mAb aggregation propensity. Different IgG1 and IgG2 molecules were tested individually and in mixtures consisting of up to four protein molecules. Antibody aggregation was induced by four different stress factors: high temperature, low pH, addition of fatty acids, and rigorous agitation. The extent of aggregation was determined from the amount of monomeric protein remaining in solution after stress. Consequently, it was possible to address the role of specific mAb regions in antibody aggregation by co-incubating Fab and Fc fragments with their respective full-length molecules. Our results revealed that the relative contribution of Fab and Fc regions in mAb aggregation is strongly dependent on pH and the stress factor applied. In addition, the CEX-based approach was used to study reversible protein precipitation due to phase separation, which demonstrated its use for a broader range of protein-protein association phenomena. In all cases, the role of Fab and Fc was clearly dissected, providing important information for engineering more stable mAb-based therapeutics.

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Identification of multiple sources of charge heterogeneity in a recombinant antibody

Cited by 481 publications

References 30 publications

Trap for MAbs: Characterization of intact monoclonal antibodies using reversed-phase HPLC on-line with ion-trap mass spectrometry

Trap for MAbs: Characterization of intact monoclonal antibodies using reversed-phase HPLC on-line with ion-trap mass spectrometry

Tissue Distribution Studies of Protein Therapeutics Using Molecular Probes: Molecular Imaging

The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

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