Comparability Testing of a Humanized Monoclonal Antibody (Synagis®) to Support Cell Line Stability, Process Validation, and Scale-Up for Manufacturing

Schenerman, Mark A.; Hope, John N.; Kletke, C.; Singh, Juspreet K.; Kimura, Roy; Tsao, Eric; Folena-Wasserman, Gail

doi:10.1006/biol.1999.0179

Cited by 56 publications

(37 citation statements)

References 26 publications

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“…), and stability profiles under accelerated and storage temperature conditions. 20,21 It has recently been shown that comparison of accelerated temperature stability profiles can be an effective and sensitive way to perform comparability assessments. 20 Because the EPD methodology provides a rapid, high throughput approach to collect and evaluate conformational stability data under accelerated conditions of temperature and pH, this approach could potentially be useful as a comparability assessment tool.…”

Section: Discussionmentioning

confidence: 99%

“…18,19 Comparability assessments in biopharmaceutical development are conducted to determine the similarities (and differences) of various preparations of a biopharmaceutical drug used in clinical development in terms of a protein drug's physicochemical properties as they relate to its safety and efficacy. 20,21 Currently, one of the major analytical challenges in this area is to identify new biophysical approaches that complement in vitro/in vivo potency assays to better assess higher order structure and conformational stability of biopharmaceutical drug candidates. Although the series of FGF-1 mutants evaluated in this study are different molecular entities, the updated EPD data analytical approach used in this study could potentially be applied to biopharmaceutical comparability assessments of different preparations of the same protein drug.…”

Section: Introductionmentioning

confidence: 99%

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An empirical phase diagram approach to investigate conformational stability of “second‐generation” functional mutants of acidic fibroblast growth factor‐1

et al. 2012

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Acidic fibroblast growth factor-1 (FGF-1) is an angiogenic protein which requires binding to a polyanion such as heparin for its mitogenic activity and physicochemical stability. To evaluate the extent to which this heparin dependence on solution stability could be reduced or eliminated, the structural integrity and conformational stability of 10 selected FGF-1 mutants were examined as a function of solution pH and temperature by a series of spectroscopic methods including circular dichroism, intrinsic and extrinsic fluorescence spectroscopy and static light scattering. The biophysical data were summarized in the form of colored empirical phase diagrams (EPDs). FGF-1 mutants were identified with stability profiles in the absence of heparin comparable to that of wild-type FGF-1 in the presence of heparin while still retaining their biological activity. In addition, a revised version of the EPD methodology was found to provide an information rich, high throughput approach to compare the effects of mutations on the overall conformational stability of proteins in terms of their response to environmental stresses such as pH and temperature.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

An empirical phase diagram approach to investigate conformational stability of “second‐generation” functional mutants of acidic fibroblast growth factor‐1

et al. 2012

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“…[39][40][41] No changes in the isoform patterns were observed due to the effect of shear ( Figure 5).…”

Section: 36mentioning

confidence: 97%

Factors influencing antibody stability at solid–liquid interfaces in a high shear environment

Biddlecombe¹,

Smith²,

Uddin³

et al. 2009

Biotechnology Progress

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A rotating disk shear device was used to study the effect of interfacial shear on the structural integrity of human monoclonal antibodies of IgG4 isotype. Factors associated with the solution conditions (pH, ionic strength, surfactant concentration, temperature) and the interface (surface roughness) were studied for their effect on the rate of IgG4 monomer loss under high shear conditions. The structural integrity of the IgG4 was probed after exposure to interfacial shear effects by SDS-PAGE, IEF, dynamic light scattering, and peptide mapping by LC-MS. This analysis revealed that the main denaturation pathway of IgG4 exposed to these effects was the formation of large insoluble aggregates. Soluble aggregation, breakdown in primary structure, and chemical modifications were not detected. The dominant factors found to affect the rate of IgG4 monomer loss under interfacial shear conditions were found to be pH and the nanometer-scale surface roughness associated with the solid-liquid interface. Interestingly, temperature was not found to be a significant factor in the range tested (15-45 degrees C). The addition of surfactant was found to have a significant stabilizing effect at concentrations up to 0.02% (w/v). Implications of these findings for the bioprocessing of this class of therapeutic protein are briefly discussed.

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“…The reason for the occurrence of this minor constituent is currently under investigation. However, sources for the heterogeneity of mAbs have been discussed elsewhere and comprise variability in PTMs introduced over the lifespan of molecules [67], the various stages in the progress of cell cultures [68], variable cultivation conditions [69], or modifications of mAbs induced by manufacturing and purification [68]. Furthermore, insufficient sub-cloning cannot be ruled out a priori.…”

Section: Characterization Of Miggs With Sds-page and Maldimentioning

confidence: 99%

Confirmation of immuno‐reactivity of the recombinant major birch pollen allergen Bet v 1a by affinity‐CIEF

et al. 2009

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Affinity-CIEF has been applied to characterize a recombinant product of the major birch pollen allergen Betula verrucosa isoform 1a (Bet v 1a) immuno-chemically. For this purpose mAbs of the IgG-type have been produced in-lab from two murine hybridoma lines, specified as clones 2 and 5.1. Both IgG clones were characterized by SDS-PAGE, MALDI-TOF-MS and CIEF. The purified IgG solutions had to be dialysed against 10 mmol/L phosphate (pH 7.4) to prevent IgG precipitation and to ensure appropriate CIEF separation. Both tested monoclonal IgGs (mIgGs) comprised four constituents covering pI ranges of 6.98-7.09 and 6.78-7.03 for clones 2 and 5.1 with major peaks at pI 7.09 and 7.03, respectively. When increasing amounts of Bet v 1a (pI 4.95) were incubated with 2.0 mumol/L mIgG, novel peaks were progressively induced in a pI range slightly more acidic than the focusing region of mIgGs. These peaks grew on the expense of original mIgG peaks. All pI values were calculated using two pI marker compounds with a repeatability of better than 0.03 units. New peaks represent complexes between Bet v 1a and mIgG either of 1:1 or of 2:1 binding stoichiometry. At a molar ratio of 2:1, saturation of both IgG paratopes with allergen (Ag) molecules was achieved as indicated by unbound Bet v 1a. The current CIEF approach addresses the proof of single epitope integrity in the course of immuno-chemical characterization of Bet v 1a. Contrary to traditional immunoassays, affinity CIEF allows for a distinction and relative quantification of mAbs, Ag-antibody complexes and Ag variants coexisting in one sample.

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Comparability Testing of a Humanized Monoclonal Antibody (Synagis®) to Support Cell Line Stability, Process Validation, and Scale-Up for Manufacturing

Cited by 56 publications

References 26 publications

An empirical phase diagram approach to investigate conformational stability of “second‐generation” functional mutants of acidic fibroblast growth factor‐1

An empirical phase diagram approach to investigate conformational stability of “second‐generation” functional mutants of acidic fibroblast growth factor‐1

Factors influencing antibody stability at solid–liquid interfaces in a high shear environment

Confirmation of immuno‐reactivity of the recombinant major birch pollen allergen Bet v 1a by affinity‐CIEF

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