Freezing and Thawing of Protein Solutions

Singh, Satish K.; Nema, Sandeep

doi:10.1002/9780470595886.ch26

Cited by 19 publications

(15 citation statements)

References 130 publications

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“…The relatively high‐protein concentrations of at least 25 mg/mL required for Raman scattering can be regarded as only a minor limitation. Proteins are usually frozen at higher concentrations because they are more stable when they are stored this way . Yet, lower protein concentrations in ice might be accessible using UV resonance Raman spectroscopy, possibly allowing a more widespread use of the Raman‐based method for protein characterization in the frozen state.…”

Section: Resultsmentioning

confidence: 99%

“…The container was equipped with cooling coils mounted in its vertical axis. Thus, high protein concentrations could be expected in half‐radial regions because of macroscopic cryoconcentration . Heat stressing of recombinant human immunoglobulin G (rhIgG)1‐A was performed for 10 min at 74°C in a volume of 1 mL on a Thermomixer Comfort by Eppendorf (Hamburg, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…Proteins are usually frozen at higher concentrations because they are more stable when they are stored this way. 1 Yet, lower protein concentrations in ice might be accessible using UV resonance Raman spectroscopy, possibly allowing a more widespread use of the Raman-based method for protein characterization in the frozen state. Signal intensities are increased between 100-and 10,000-fold by using excitation wavelengths that are absorbed by the protein.…”

Section: Potential Methods Limitationsmentioning

confidence: 99%

“…Freezing bulk volumes of pharmaceutical protein solutions is a widely used, yet potentially critical unit operation. Increased shelf‐life and flexibility in storage and logistics is opposed by the risk of inactivation and aggregation of considerable amounts of high‐value protein drugs upon freezing and thawing (F/T) . Protein degradation can be triggered by cold denaturation, cryoconcentration of (co)solutes, and concomitant reduction of solubility as well as by exposure to surfaces of ice crystals or precipitated or crystallized excipients .…”

Section: Introductionmentioning

confidence: 99%

“…Increased shelf-life and flexibility in storage and logistics is opposed by the risk of inactivation and aggregation of considerable amounts of high-value protein drugs upon freezing and thawing (F/T). 1 Protein degradation can be triggered by cold denaturation, cryoconcentration of (co)solutes, and concomitant reduction of solubility as well as by exposure to surfaces of ice crystals or precipitated or crystallized excipients. 2 Given the large volumes of up to several hundred liters of concentrated protein product stored at industrial scale, any information about protein integrity inside a freeze container is highly desirable and would provide important information for F/T process design.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

In Situ Protein Secondary Structure Determination in Ice: Raman Spectroscopy-Based Process Analytical Tool for Frozen Storage of Biopharmaceuticals

Roessl

Leitgeb

Pieters³

et al. 2014

Journal of Pharmaceutical Sciences

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Potential Methods Limitationsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

In Situ Protein Secondary Structure Determination in Ice: Raman Spectroscopy-Based Process Analytical Tool for Frozen Storage of Biopharmaceuticals

Roessl

Leitgeb

Pieters³

et al. 2014

Journal of Pharmaceutical Sciences

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Design of experiments reveals critical parameters for pilot‐scale freeze‐and‐thaw processing of L‐lactic dehydrogenase

Roessl

Humi

Leitgeb

et al. 2015

Biotechnology Journal

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Freezing constitutes an important unit operation of biotechnological protein production. Effects of freeze-and-thaw (F/T) process parameters on stability and other quality attributes of the protein product are usually not well understood. Here a design of experiments (DoE) approach was used to characterize the F/T behavior of L-lactic dehydrogenase (LDH) in a 700-mL pilot-scale freeze container equipped with internal temperature and pH probes. In 24-hour experiments, target temperature between -10 and -38°C most strongly affected LDH stability whereby enzyme activity was retained best at the highest temperature of -10°C. Cooling profile and liquid fill volume also had significant effects on LDH stability and affected the protein aggregation significantly. Parameters of the thawing phase had a comparably small effect on LDH stability. Experiments in which the standard sodium phosphate buffer was exchanged by Tris-HCl and the non-ionic surfactant Tween 80 was added to the protein solution showed that pH shift during freezing and protein surface exposure were the main factors responsible for LDH instability at the lower freeze temperatures. Collectively, evidence is presented that supports the use of DoE-based systematic analysis at pilot scale in the identification of F/T process parameters critical for protein stability and in the development of suitable process control strategies.

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Raman spectroscopy as a process analytical technology to investigate biopharmaceutical freeze concentration processes

Weber

Hubbuch

2021

Biotech & Bioengineering

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Freezing processes are a well-established unit operation in the biopharmaceutical industry to increase the shelf-life of protein-based drugs. While freezing reduces degradation reaction rates, it may also exert stresses such as freeze concentration.Macroscopic freeze concentration in large-scale freezing processes has been described thoroughly by examination of frozen bulk material, but the transient process leading to such freeze concentration profiles has not been monitored yet for biopharmaceutical solutions. In this study, Raman spectroscopy as a process analytical technology is demonstrated for model formulations containing monoclonal antibodies (mAbs) or bovine serum albumin (BSA) in varying concentrations of sucrose and buffer salts. Therefore, a Raman probe was immersed into a bulk volume at different heights, monitoring the freeze concentration in the liquid phase during the freezing processes. Partial least square regression models were used to quantitatively discriminate between the protein and excipients simultaneously. The freeze concentration profiles were dependend on freezing temperature and formulation with freeze concentrations up to 2.4-fold. Convection currents at the bottom of the freezing container were observed with a maximum height of 1 mm. Furthermore, freeze concentration was correlated with the sucrose concentration in a formulation.Analysis of the freeze concentration slope indicated diffusion from the bottom to the top of the container. In summary, Raman spectroscopy is a valuable tool for process validation of freeze concentration simulations and to overcome scale-dependent challenges.

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Freezing and Thawing of Protein Solutions

Cited by 19 publications

References 130 publications

In Situ Protein Secondary Structure Determination in Ice: Raman Spectroscopy-Based Process Analytical Tool for Frozen Storage of Biopharmaceuticals

In Situ Protein Secondary Structure Determination in Ice: Raman Spectroscopy-Based Process Analytical Tool for Frozen Storage of Biopharmaceuticals

Design of experiments reveals critical parameters for pilot‐scale freeze‐and‐thaw processing of L‐lactic dehydrogenase

Raman spectroscopy as a process analytical technology to investigate biopharmaceutical freeze concentration processes

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