Freeze concentration during freezing: How does the maximally freeze concentrated solution influence protein stability?

Seifert, Ivonne; Frieß, Wolfgang

doi:10.1016/j.ijpharm.2020.119810

Cited by 10 publications

(4 citation statements)

References 49 publications

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“…In contrast, when samples are stored above T g ′, the viscosity drops, facilitating irreversible denaturation and aggregation ( Franks, 1998 ). Above T g ′, the viscosity of the FCM decreases exponentially with increasing temperature ( Seifert and Friess, 2020 ). The lower viscosity is associated with increased molecular mobility and consequently accelerated physical and chemical instability ( Pansare and Patel, 2016 ).…”

Section: Discussionmentioning

confidence: 99%

The effect of mAb and excipient cryoconcentration on long-term frozen storage stability – Part 1: Higher molecular weight species and subvisible particle formation

Bluemel

Anuschek

Buecheler

et al. 2022

International Journal of Pharmaceutics: X

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Cryoconcentration upon large-scale freezing of monoclonal antibody (mAb) solutions leads to regions of different ratios of low molecular weight excipients, like buffer species or sugars, to protein. This study focused on the impact of the buffer species to mAb ratio on aggregate formation after frozen storage at −80 °C, −20 °C, and − 10 °C after 6 weeks, 6 months, and 12 months. An optimised sample preparation was established to measure T g ′ of samples with different mAb to histidine ratios via differential scanning calorimetry (DSC). After storage higher molecular weight species (HMWS) and subvisible particles (SVPs) were detected using size-exclusion chromatography (SEC) and FlowCam, respectively. For all samples, sigmoidal curves in DSC thermograms allowed to precisely determine T g ′ in formulations without glass forming sugars. Storage below T g ′ did not lead to mAb aggregation. Above T g ′, at −20 °C and − 10 °C, small changes in mAb and buffer concentration markedly impacted stability. Samples with lower mAb concentration showed increased formation of HMWS. In contrast, higher concentrated samples led to more SVPs. A shift in the mAb to histidine ratio towards mAb significantly increased overall stability. Cryoconcentration upon large-scale freezing affects mAb stability, although relative changes compared to the initial concentration are small. Storage below T g ′ completely prevents mAb aggregation and particle formation.

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Section: Discussionmentioning

confidence: 99%

The effect of mAb and excipient cryoconcentration on long-term frozen storage stability – Part 1: Higher molecular weight species and subvisible particle formation

Bluemel

Anuschek

Buecheler

et al. 2022

International Journal of Pharmaceutics: X

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“…[37] Furthermore, recrystallization can also trigger the creation of additional interfaces where proteins may adsorb, resulting in denaturation. [38] The increase in salt content in the surrounding media because of freeze concentration is another significant factor in protein instability in addition to damage caused by ice recrystallization [39] ; freeze concentration occurs when the freezing process causes solutes to be excluded from the solution because of the creation of ice, resulting in a higher solute concentration in the residual solution. Notably, PLLSA exhibits strong intermolecular interactions that can prevent osmotic shock by trapping salt and water in the molecules (by forming a reversible matrix), consequently lowering the freezing concentration.…”

Section: Protein Protection Studiesmentioning

confidence: 99%

Polyampholyte‐Based Polymer Hydrogels for the Long‐Term Storage, Protection and Delivery of Therapeutic Proteins

Rajan

Kumar

Zhao

et al. 2023

Adv Healthcare Materials

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Protein storage and delivery are crucial for biomedical applications such as protein therapeutics and recombinant proteins. Lack of proper protocols results in the denaturation of proteins, rendering them inactive and manifesting undesired side effects. In this study, polyampholyte-based (succinylated 𝝐-poly-l-lysine) hydrogels containing polyvinyl alcohol and polyethylene glycol polymer matrices to stabilize proteins are developed. These hydrogels facilitated the loading and release of therapeutic amounts of proteins and withstood thermal and freezing stress (15 freeze-thaw cycles and temperatures of −80 °C and 37 °C), without resulting in protein denaturation and aggregation. To the best of our knowledge, this strategy has not been applied to the design of hydrogels constituting polymers, (in particular, polyampholyte-based polymers) which have inherent efficiency to stabilize proteins and protect them from denaturation. Our findings can open up new avenues in protein biopharmaceutics for the design of materials that can store therapeutic proteins long-term under severe stress and safely deliver them.

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“…In addition to ice recrystallization damage, another major cause of protein instability is the crystallization of water to ice during freezing, causing an increase in salt concentration in the surrounding medium because of freeze concentration . Freeze concentration in turn is a phenomenon where the freezing process results in the exclusion of solutes from the solution due to the formation of ice, resulting in higher solute concentration in the remaining solution .…”

Section: Introductionmentioning

confidence: 99%

“…29 In addition to ice recrystallization damage, another major cause of protein instability is the crystallization of water to ice during freezing, causing an increase in salt concentration in the surrounding medium because of freeze concentration. 30 Freeze concentration in turn is a phenomenon where the freezing process results in the exclusion of solutes from the solution due to the formation of ice, resulting in higher solute concentration in the remaining solution. 12 To this end, Matsumura and his group has recently established that succinylated ε-poly-L-lysine (PLLSA), which is a polyampholyte, possesses strong intermolecular interactions, which can reduce the osmotic damage to decrease the freeze concentration by trapping salt in the molecules.…”

Section: ■ Introductionmentioning

confidence: 99%

Design of an Ice Recrystallization-Inhibiting Polyampholyte-Containing Graft Polymer for Inhibition of Protein Aggregation

2021

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Freezing-induced damage to proteins, through osmotic stress and ice recrystallization, during protein processing and long-term storage is a serious concern and may lead to loss of protein activity owing to denaturation. In this study, graft copolymers composed of a cryoprotective polymer (capable of preventing osmotic stress) and poly(vinyl alcohol) (PVA; known for its high ice recrystallization inhibition (IRI) property) were developed. The polymers had high IRI activity, albeit slightly lower than that of PVA alone, but substantially higher than that of succinylated ε-poly-l-lysine (PLLSA) alone. The graft polymers showed an efficiency higher than that of PVA or PLLSA alone in protecting proteins from multiple freeze–thaw cycles, as well as during prolonged freezing, indicating a synergy between PVA and PLLSA. The PLLSA-based graft polymer is a promising material for use in protein biopharmaceutics for the long-term storage of proteins under freezing conditions.

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Freeze concentration during freezing: How does the maximally freeze concentrated solution influence protein stability?

Cited by 10 publications

References 49 publications

The effect of mAb and excipient cryoconcentration on long-term frozen storage stability – Part 1: Higher molecular weight species and subvisible particle formation

The effect of mAb and excipient cryoconcentration on long-term frozen storage stability – Part 1: Higher molecular weight species and subvisible particle formation

Polyampholyte‐Based Polymer Hydrogels for the Long‐Term Storage, Protection and Delivery of Therapeutic Proteins

Design of an Ice Recrystallization-Inhibiting Polyampholyte-Containing Graft Polymer for Inhibition of Protein Aggregation

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