Freeze-concentration of solutes during bulk freezing and its impact on protein stability

Minatovicz, Bruna; Sun, Lanxiang; Foran, Christopher; Chaudhuri, Bodhisattwa; Tang, Charlie; Shameem, Mohammed

doi:10.1016/j.jddst.2020.101703

Cited by 17 publications

(6 citation statements)

References 39 publications

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“…During the freezing process, the ice formation excludes solutes (including protein) from the growing ice crystal. In general, solutes concentrate towards the center and bottom during freezing [4]. This concentration effect leads to other effects that induce the denaturation of the protein.…”

Section: Cryoconcentrationmentioning

confidence: 99%

See 1 more Smart Citation

Accomplishing Scalability by Using Plate‐Based Freeze and Thaw Technologies

Jenewein¹,

Breitrainer²

2022

Chemie Ingenieur Technik

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Controlling the freezing behavior of bulk drug substance opens doors to process reproducibility and consistent quality of the final drug product by maintaining uniform conditions for the biopharmaceuticals during freezing, and consequently for frozen storage and shipment. This simplifies the commercial bulk production, particularly when manufacturing different drug substances at different sensitivities and temperature set points and enables scalability. The leverage of the ice front growth speed had a significant impact on controllability and, as a result, on the protein quality.

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Section: Cryoconcentrationmentioning

confidence: 99%

“…The freezing rate was shown to have significant impact on the protein stability during bulk freezing [4]. In most cases, a low freezing rate causes the ice to form slowly enough for the proteins to be pushed along the ice rather than being trapped by the ice front of bags (see Fig.…”

Section: Freezing Ratementioning

confidence: 99%

Accomplishing Scalability by Using Plate‐Based Freeze and Thaw Technologies

Jenewein¹,

Breitrainer²

2022

Chemie Ingenieur Technik

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“…3). These variable freezing rates and ice front velocities at different regions within the bottles can contribute to the redistribution of solutes and substantial heterogeneity in the composition of the frozen bulk, which can foster protein destabilization (9,23).…”

Section: Effect Of the Processing Conditions On F/t Ratesmentioning

confidence: 99%

Use of a Design of Experiments (DoE) Approach to Optimize Large-Scale Freeze-Thaw Process of Biologics

2021

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Large volumes of protein solutions are commonly stored in a frozen state before further drug product fill and finish. This study aimed to establish a design space to perform large-scale freeze-thaw (F/T) processes of biotherapeutics without inducing protein destabilization. A response surface model was designed to evaluate the following main factors and interactions: fill volume of the protein solution in 1-L containers, distance among nine containers during both F/T, freezer set temperature, and a novel forced air flow methodology during thawing. The analysis from 46 experimental runs indicated over 4-fold increase in the freezing rate by lowering the freezing temperature from −20 to −80°C, and the forced air flow at 98 fpm doubled the thawing rate. Furthermore, multivariate linear regression modeling revealed the significant impact of all main factors investigated on lactate dehydrogenase (LDH) quality attributes. The factor that most strongly affected the retention of LDH activity was the loading distance: ≥ 5 cm among containers positively affected the LDH activity response in 50.6%. The factor that most strongly retained the LDH tetramers was the set freezer temperature towards the lower range of −80°C (2.2% higher tetramer retention compared to −20°C freezing, due to faster freezing rate). In summary, this DoEbased systematic analysis increased F/T process understanding at large scale, identified critical F/T process parameters, and confirmed the feasibility of applying faster freezing and forced air thawing procedures to maintain the stability of LDH solutions subject to largescale F/T.

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“…Thus, ice crystallization leads to, among other things, a pronounced increase in ionic strength and viscosity. The ice crystallization and cryo-concentration induce stresses which can lead to protein aggregation. , Additionally, there is a large body of evidence suggesting loss in protein activity due to its adsorption at the hydrophobic interfaces generated at the ice–water and ice–air interfaces . In most investigations, the protein stability (specifically aggregation and activity) was evaluated before and after one or multiple (up to five) freeze–thaw cycles.…”

Section: Introductionmentioning

confidence: 99%

“…6,10 Additionally, there is a large body of evidence suggesting loss in protein activity due to its adsorption at the hydrophobic interfaces generated at the ice−water and ice−air interfaces. 11 In most investigations, the protein stability (specifically aggregation and activity) was evaluated before and after one or multiple (up to five) freeze−thaw cycles. Similar studies have also been conducted after the entire freeze-drying cycle by reconstituting the lyophile.…”

Section: ■ Introductionmentioning

confidence: 99%

Reversible Self-Association in Lactate Dehydrogenase during Freeze–Thaw in Buffered Solutions Using Neutron Scattering

et al. 2021

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The aims of this work were to evaluate the effect of freezing and thawing stresses on lactate dehydrogenase (LDH) stability under three conditions. (i) In a solution buffered with sodium phosphate (NaP; 10 and 100 mM). The selective crystallization of disodium hydrogen phosphate during freezing caused a pronounced pH shift. (ii) In a solution buffered with histidine, where there was no pH shift due to buffer salt crystallization. (iii) At different concentrations of LDH so as to determine the self-stabilizing ability of LDH. The change in LDH tetrameric conformation was measured by small-angle neutron scattering (SANS). The pH of the phosphate buffer solutions was monitored as a function of temperature to quantify the pH shift. The conditions of buffer component crystallization from solution were identified using low-temperature X-ray diffractometry. Dynamic light scattering (DLS) enabled us to determine the effect of freeze-thawing on the protein aggregation behavior. LDH, at a high concentration (1000 μg/mL; buffer concentration 10 mM), has a pronounced self-stabilizing effect and did not aggregate after five freeze–thaw cycles. At lower LDH concentrations (10 and 100 μg/mL), only with the selection of an appropriate buffer, irreversible aggregation could be avoided. While SANS provided qualitative information with respect to protein conformation, the insights from DLS were quantitative with respect to the particle size of the aggregates. SANS is the only technique which can characterize the protein both in the frozen and thawed states.

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Freeze-concentration of solutes during bulk freezing and its impact on protein stability

Cited by 17 publications

References 39 publications

Accomplishing Scalability by Using Plate‐Based Freeze and Thaw Technologies

Accomplishing Scalability by Using Plate‐Based Freeze and Thaw Technologies

Use of a Design of Experiments (DoE) Approach to Optimize Large-Scale Freeze-Thaw Process of Biologics

Reversible Self-Association in Lactate Dehydrogenase during Freeze–Thaw in Buffered Solutions Using Neutron Scattering

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