Frozen State Storage Instability of a Monoclonal Antibody: Aggregation as a Consequence of Trehalose Crystallization and Protein Unfolding

Singh, Satish K.; Kolhe, Parag; Mehta, Anjali; Chico, Steven; Lary, Alanta; Huang, Min

doi:10.1007/s11095-010-0343-z

Cited by 110 publications

(73 citation statements)

References 22 publications

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“…24 Compact dimers2 of mAb_A and mAb_B were identified in bulk material stored for a long time at ¡20 C. During freezing, local elevated concentrations of the mAb itself or of excipients, salts or buffer components might induce protein aggregation as described in Liu et al 27 Furthermore, the storage temperature of ¡20 C might favor the crystallization of trehalose present in formulation buffer out of a supersaturated state, which has been shown to accelerate aggregate formation. 28,29 However, also other unidentified factors might influence the formation of compact dimers.…”

Section: Discussionmentioning

confidence: 99%

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

Plath

Ringler

Graff-Meyer

et al. 2016

mAbs

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(2016) Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs, mAbs, 8:5, 928-940, DOI: 10.1080/19420862.2016 To link to this article: https://doi.org/10. 1080/19420862.2016 ABSTRACTThe formation of undesired high molecular weight species such as dimers is an important quality attribute for therapeutic monoclonal antibody formulations. Therefore, the thorough understanding of mAb dimerization and the detailed characterization mAb dimers is of great interest for future pharmaceutical development of therapeutic antibodies. In this work, we focused on the analyses of different mAb dimers regarding size, surface properties, chemical identity, overall structure and localization of possible dimerization sites. Dimer fractions of different mAbs were isolated to a satisfactory purity from bulk material and revealed 2 predominant overall structures, namely elongated and compact dimer forms. The elongated dimers displayed one dimerization site involving the tip of the Fab domain. Depending on the stress applied, these elongated dimers are connected either covalently or non-covalently. In contrast, the compact dimers exhibited non-covalent association. Several interaction points were detected for the compact dimers involving the hinge region or the base of the Fab domain. These results indicate that mAb dimer fractions are rather complex and may contain more than one kind of dimer. Nevertheless, the overall appearance of mAb dimers suggests the existence of 2 predominant dimeric structures, elongated and compact, which are commonly present in preparations of therapeutic mAbs.

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Section: Discussionmentioning

confidence: 99%

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

Plath

Ringler

Graff-Meyer

et al. 2016

mAbs

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“…Furthermore, the denaturation of protein during freezing could also occur as a consequence of changes in the solute concentration due to ice formation, exposure of protein to the ice-liquid interface and changes of the pH value surrounding the protein [11]. Freeze-thaw cycles are routinely used as a tool to determine the combined effects of these different freezing related stresses on protein stability [11,12].…”

Section: Introductionmentioning

confidence: 99%

Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life

Rašković

Vatić

Anđelković

et al. 2016

Biochemical Engineering Journal

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“…Further, the denaturation of a protein during freezing could also occur as a consequence of changes in solute concentration due to ice formation and changes of pH value in the protein surrounding [15]. Freeze-thaw cycles are 4 routinely used as a tool to determine the combined effects of these different freezing related stresses on protein stability [15,16].…”

Section: Introductionmentioning

confidence: 99%

Fourier transform infrared spectroscopy provides an evidence of papain denaturation and aggregation during cold storage

Rašković

Popović

Ostojić

et al. 2015

Spectrochimica Acta Part A: Molecular and Biomolecular Spectros

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Papain is a cysteine protease with wide substrate specificity and many applications. Despite its widespread applications, cold stability of papain has never been studied. Here, we used differential spectroscopy to monitor thermal denaturation process. Papain was the most stabile from 45 °C to 60 °C with ΔG°321 of 13.9±0.3 kJ/mol and Tm value of 84±1 °C. After cold storage, papain lost parts of its native secondary structures elements which gave an increase of 40% of intermolecular β-sheet content (band maximum detected at frequency of 1621 cm(-1) in Fourier transform infrared (FT-IR) spectrum) indicating the presence of secondary structures necessary for aggregation. The presence of protein aggregates after cold storage was also proven by analytical size exclusion chromatography. After six freeze-thaw cycles around 75% of starting enzyme activity of papain was lost due to cold denaturation and aggregation of unfolded protein. Autoproteolysis of papain did not cause significant loss of the protein activity. Upon the cold storage, papain underwent structural rearrangements and aggregation that correspond to other cold denatured proteins, rather than autoproteolysis which could have the commercial importance for the growing polypeptide based industry.

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Frozen State Storage Instability of a Monoclonal Antibody: Aggregation as a Consequence of Trehalose Crystallization and Protein Unfolding

Abstract: Aggregation in the frozen state when stored above the glass transition temperature is a consequence of balance between rate of crystallization leading to loss of cryoprotectant, rate of aggregation of the unfolded protein molecules, and rate of refolding that prevents aggregation.

Cited by 110 publications

References 22 publications

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life

Fourier transform infrared spectroscopy provides an evidence of papain denaturation and aggregation during cold storage

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