Effects of Solution Conditions, Processing Parameters, and Container Materials on Aggregation of a Monoclonal Antibody during Freeze-Thawing

Kueltzo, Lisa A.; Wang, W.e.i.; Randolph, Theodore W.; Carpenter, John F.

doi:10.1002/jps.21110

Cited by 178 publications

(160 citation statements)

References 22 publications

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“…1 As with any protein drug, monoclonal antibodies can undergo a variety of degradation pathways. [2][3][4] One of the most common routes of instability is the formation of antibody oligomers and higher molecular weight aggregates. [3][4][5][6] Antibody aggregation can pose a significant challenge during drug development, storage, and delivery.…”

Section: Introductionmentioning

confidence: 99%

“…[2][3][4] One of the most common routes of instability is the formation of antibody oligomers and higher molecular weight aggregates. [3][4][5][6] Antibody aggregation can pose a significant challenge during drug development, storage, and delivery. 4 Such instability can impact the ability to consistently manufacture the high purity drug product.…”

Section: Introductionmentioning

confidence: 99%

“…High molecular weight (HMW) species formation has been described for antibodies under a variety of circumstances, including freeze/thaw, 3,6,9 agitation, 9 and thermal stress. 5,6,9 Additionally, under physiological conditions, antibody light chains and associated variable light chains have been shown to possess the ability to form amyloid, and have been implicated in free light chain amyloidosis, a neurodegenerative disorder.…”

Section: Introductionmentioning

confidence: 99%

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Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregates

et al. 2010

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Aggregation of human therapeutic antibodies represents a significant hurdle to product development. In a test across multiple antibodies, it was observed that IgG1 antibodies aggregated less, on average, than IgG2 antibodies under physiological pH and mildly elevated temperature. This phenomenon was also observed for IgG1 and IgG2 subclasses of anti-streptavidin, which shared 95% sequence identity but varied in interchain disulfide connectivity. To investigate the structural and covalent changes associated with greater aggregation in IgG2 subclasses, soluble aggregates from the two forms of anti-streptavidin were isolated and characterized. Sedimentation velocity analytical ultracentrifugation (SV-AUC) measurements confirmed that the aggregates were present in solution, and revealed that the IgG1 aggregate was composed of a predominant species, whereas the IgG2 aggregate was heterogeneous. Tertiary structural changes accompanied antibody aggregation as evidenced by greater ANS (8-Anilino-1-naphthalene sulfonic acid) binding to the aggregates over monomer, and differences in disulfide character and tryptophan environments between monomer, oligomer and aggregate species, as observed by near-UV circular dichroism (CD). Differences between subclasses were observed in the secondary structural changes that accompanied aggregation, particularly in the intermolecular b-sheet and turn structures between the monomer and aggregate species. Free thiol determination showed 2.4-fold lower quantity of free cysteines in the IgG1 subclass, consistent with the 2.4-fold reduction in aggregation of the IgG1 form when compared with IgG2 under these conditions. These observations suggested an important role for disulfide bond formation, as well as secondary and tertiary structural transitions, during antibody aggregation. Such degradations may be minimized using appropriate formulation conditions.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregates

et al. 2010

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“…To characterize the loss of function due to cold temperatures, we examined the effect of repeated freeze-thaw cycles on the function of these proteins in a manner similar to the method that Kueltzo et al [20] applied for studying the aggregation of a monoclonal antibody. Our results affirm the starting hypothesis that IDPs, in contrast with globular proteins, are resistant to cold treatment.…”

Section: Introductionmentioning

confidence: 99%

Cold stability of intrinsically disordered proteins

2008

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Edited by Miguel De la Rosa Keywords:Cold denaturation Cold shock protein LEA Intrinsically disordered protein Chaperone Stability a b s t r a c t Contrary to globular proteins, intrinsically disordered proteins (IDPs) lack a folded structure and they do not lose solubility at elevated temperatures. Although this should also be true at low temperatures, cold stability of IDPs has not been addressed in any scientific work so far. As direct characterization of cold-denaturation is difficult, we approached the problem through a freezinginduced loss-of-function model of globular-disordered functional protein pairs (m-calpain-calpastatin, tubulin-Map2c, Hsp90-ERD14). Our results affirm that in contrast with globular proteins IDPs are resistant to cold treatment. The theoretical and functional aspects of this observation are discussed.

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“…[4][5][6] These external stressors include temperature changes, lyophilization, rehydration, oxidation, and light exposure. [5][6][7] Interactions between proteins and foreign materials, such as rubber, metal particles, and administration devices, can also occur. [8][9][10][11][12] For injectable formulations, syringes (glass or polypropylene) and needles are used for reconstitution and administration.…”

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confidence: 99%

Reconstitution of L-Asparaginase in Siliconized Syringes with Shaking and Headspace Air Induces Protein Aggregation

Uchino

Miyazaki

Ohkawa

et al. 2015

CHEMICAL & PHARMACEUTICAL BULLETIN

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The aim of this study was to characterize protein aggregation during reconstitution of a highly concentrated solution of lyophilized L-asparaginase (L-ASP). The effect of the preparation method on L-ASP aggregation using siliconized or non-siliconized syringes and the effect of storage after preparation were evaluated by far-UV circular dichroism spectroscopy, Raman microscopy, flow cytometry, and flow particle image analysis. To investigate the effect of syringe type in combination with shaking and headspace air on L-ASP aggregation, four kinds of L-ASP in 5% glucose solutions were prepared (in the presence or absence of silicon oil and headspace air). Slight differences in L-ASP secondary structure were observed between the siliconized and non-siliconized syringe systems before shaking. Large numbers of sub-visible (0.1-100 µm) and submicron (0.1-1 µm) particles were formed by preparation with siliconized syringes and the combination of shaking and headspace air. The number of aggregated particles was not decreased with increased storage time. The Raman microscopy, flow cytometry and flow particle image results suggested that L-ASP interacted with silicone oil, which induced aggregation. Nevertheless, sub-visible and submicron particles were also formed with non-siliconized syringes. However, using non-siliconized syringes, the number of aggregated particles decreased with storage. No changes in particle character were observed before or after shaking with headspace air in non-siliconized syringes, indicating that soluble aggregates formed and dissolved with storage. Silicone oil in syringes, in combination with shaking and headspace air, strongly affected the aggregation of lyophilized L-ASP formulations during preparation.Key words protein aggregation; silicone oil; L-asparaginase (L-ASP); sub-visible particle; flow cytometry; Raman microscopy Various therapeutic proteins have been commercialized owing to recent advances in biotechnology and formulation. These therapeutic proteins are used for the treatment of numerous diseases including cancer, autoimmune diseases, and diabetes mellitus. However, it has been reported that protein aggregates in these products have caused side effects, immunogenicity, and allergic reactions.1-3) Substantial effort has been expended to develop stable formulations to prevent these events. These stable formulations are necessary for safe and effective pharmaceutical therapies.There has been great interest in the external stress affecting protein stability during production, filling, shipment, storage, and final administration. [4][5][6] These external stressors include temperature changes, lyophilization, rehydration, oxidation, and light exposure. [5][6][7] Interactions between proteins and foreign materials, such as rubber, metal particles, and administration devices, can also occur. [8][9][10][11][12] For injectable formulations, syringes (glass or polypropylene) and needles are used for reconstitution and administration. In these devices, silicone oil is used as a lubricant ...

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Effects of Solution Conditions, Processing Parameters, and Container Materials on Aggregation of a Monoclonal Antibody during Freeze-Thawing

Cited by 178 publications

References 22 publications

Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregates

Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregates

Cold stability of intrinsically disordered proteins

Reconstitution of L-Asparaginase in Siliconized Syringes with Shaking and Headspace Air Induces Protein Aggregation

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