Protein Aggregation in Frozen Trehalose Formulations: Effects of Composition, Cooling Rate, and Storage Temperature

Connolly, Brian D.; Le, Lan Thi Ha; Patapoff, Thomas W.; Cromwell, Mary; Moore, Jamie; Lam, Philippe

doi:10.1002/jps.24646

Cited by 64 publications

(38 citation statements)

References 50 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

View full text Add to dashboard Cite

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“…Connolly et al 25 studied aggregation of mAb in the frozen state with 3 different excipients (sucrose, trehalose, and mannitol) during storage in the frozen state. Elevated levels of protein aggregates were observed after 28 days of storage at À20 C for 2 formulations, trehalose-and mannitol-based, whereas the sucrose-based formulation remained stable.…”

Section: Instability Of Protein Formulations During Freeze Storage: Examplesmentioning

confidence: 99%

Freezing of Biologicals Revisited: Scale, Stability, Excipients, and Degradation Stresses

Authelin

Rodrigues

Tchessalov

et al. 2020

Journal of Pharmaceutical Sciences

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Although many biotech products are successfully stored in the frozen state, there are cases of degradation of biologicals during freeze storage. These examples are discussed in the Perspective to emphasize the fact that stability of frozen biologicals should not be taken for granted. Frozen-state degradation (predominantly, aggregation) has been linked to crystallization of a cryoprotector in many cases. Other factors, for example, protein unfolding (either due to cold denaturation or interaction of protein molecules with ice crystals), could also contribute to the instability. As a hypothesis, additional freezingrelated destabilization pathways are introduced in the paper, that is, air bubbles formed on the ice crystallization front, and local pressure and mechanical stresses due to volume expansion during waterto-ice transformation. Furthermore, stability of frozen biologicals can depend on the sample size, via its impact on the freezing kinetics (i.e., cooling rates and freezing time) and cryoconcentration effects, as well as on the mechanical stresses associated with freezing. We conclude that, although fundamentals of freezing processes are fairly well described in the current literature, there are important gaps to be addressed in both scientific foundations of the freezing-related manufacturing processes and implementation of the available knowledge in practice.

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“…1) as shown in freeze-drying studies. 44,52 Thermal history (such as annealing or the freeze-drying or spray-drying condition) affects immiscibility differently through changing component mobility. 7 Annealing in freeze-drying which raises product temperature above the Tg 0 (the glass transition temperature of the maximally freeze-concentrated solutions) to produce a good cake structure can introduce sufficient molecular mobility to solutes thereby promoting phase separation.…”

Section: Immiscibility Between Componentsmentioning

confidence: 99%

“…Trehalose crystallization can be facilitated by crystallizing solutes such as mannitol while limited by non-crystallizing components like sucrose or protein. 52,81 This crystallization behavior of trehalose might be due to the lower solubility, hygroscopicity, and ability to form clusters at higher concentrations compared to other saccharides like sucrose. 82 Despite the complex crystallization process and transformation between different crystal forms, the dihydrate crystal reported in the study above returns to the amorphous form instead of transforming into the anhydrous crystal.…”

Section: Crystallization Of Componentsmentioning

confidence: 99%

“…1,30,[146][147][148][149] Trehalose crystallization after freeze-drying, even followed by subsequent amorphization, increased the amount of aggregated monoclonal antibody regardless of the amorphous state. 52,150 After spray-drying or freezedrying, amorphous stabilizers can crystallize after exposure to high relative humidities and relatively high temperatures. 147,151 Processing of an amorphous product can also introduce stabilizer crystallization such as in the case of compaction-induced crystallization of trehalose in freeze-dried trehalose-alkaline phosphatase.…”

Section: Destabilization Caused By Excipient Crystallizationmentioning

confidence: 99%

See 1 more Smart Citation

Inhomogeneous Distribution of Components in Solid Protein Pharmaceuticals: Origins, Consequences, Analysis, and Resolutions

Nguyen¹,

Frijlink²,

Hinrichs³

2020

Journal of Pharmaceutical Sciences

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Successful development of stable solid protein formulations usually requires the addition of one or several excipients to achieve optimal stability. In these products, there is a potential risk of an inhomogeneous distribution of the various ingredients, specifically the ratio of protein and stabilizer may vary. Such inhomogeneity can be detrimental for stability but is mostly neglected in literature. In the past, it was challenging to analyze inhomogeneous component distribution, but recent advances in analytical techniques have revealed new options to investigate this phenomenon. This paper aims to review fundamental aspects of the inhomogeneous distribution of components of freeze-dried and spray-dried protein formulations. Four key topics will be presented and discussed, including the sources of component inhomogeneity, its consequences on protein stability, the analytical methods to reveal component inhomogeneity, and possible solutions to prevent or mitigate inhomogeneity.

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Protein Aggregation in Frozen Trehalose Formulations: Effects of Composition, Cooling Rate, and Storage Temperature

Cited by 64 publications

References 50 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

Freezing of Biologicals Revisited: Scale, Stability, Excipients, and Degradation Stresses

Inhomogeneous Distribution of Components in Solid Protein Pharmaceuticals: Origins, Consequences, Analysis, and Resolutions

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