Liquid-liquid phase separation of a monoclonal antibody at low ionic strength: Influence of anion charge and concentration

Reiche, Katharina; Hartl, Josef; Blume, Alfred; Garidel, Patrick

doi:10.1016/j.bpc.2016.08.003

Cited by 46 publications

(58 citation statements)

References 89 publications

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“…[89][90][91][92][93][94] The addition of (chaotropic) salts 86,95,96 or charged excipients, for example, amino acids with a hydrophobic moiety such as histidine and arginine 96,97 are reported to lower the tendency to form associates, which in turn also lowers the viscosity of the solution. Similarly, reversible association that leads to liquidliquid phase separation was recently reported to follow similar trends 92,[98][99][100][101] ; this is seen especially at low temperatures, for example, during intermediate storage during DSP. Often a balance in excipient concentrations between high repulsive net charge at low ionic strength and negative effects from screened charges at high ionic strength needs to be established experimentally.…”

Section: Purification/chromatographic Steps: Impact Of Salt and Ionicsupporting

confidence: 55%

Stress Factors in mAb Drug Substance Production Processes: Critical Assessment of Impact on Product Quality and Control Strategy

Das

Narhi

Sreedhara³

et al. 2020

Journal of Pharmaceutical Sciences

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The success of biotherapeutic development heavily relies on establishing robust production platforms. During the manufacturing process, the protein is exposed to multiple stress conditions that can result in physical and chemical modifications. The modified proteins may raise safety and quality concerns depending on the nature of the modification. Therefore, the protein modifications potentially resulting from various process steps need to be characterized and controlled. This commentary brings together expertise and knowledge from biopharmaceutical scientists and discusses the various manufacturing process steps that could adversely impact the quality of drug substance (DS). The major process steps discussed here are commonly used in mAb production using mammalian cells. These include production cell culture, harvest, antibody capture by protein A, virus inactivation, polishing by ion-exchange chromatography, virus filtration, ultrafiltration-diafiltration, compounding followed by release testing, transportation and storage of final DS. Several of these process steps are relevant to protein DS production in general. The authors attempt to critically assess the level of risk in each of the DS processing steps, discuss strategies to control or mitigate protein modification in these steps, and recommend mitigation approaches including guidance on development studies that mimic the stress induced by the unit operations.

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Section: Purification/chromatographic Steps: Impact Of Salt and Ionicsupporting

confidence: 55%

Stress Factors in mAb Drug Substance Production Processes: Critical Assessment of Impact on Product Quality and Control Strategy

Das

Narhi

Sreedhara³

et al. 2020

Journal of Pharmaceutical Sciences

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show abstract

“…Therefore, all mAb drug candidates should undergo characterization of their physicochemical properties in order to mitigate the risks of significant instability, and this should be performed at the front end of the development process or during a discovery phase to reduce drug development costs [ 7 , 8 ]. One of the key risks is the loss of colloidal stability, specifically undesirable liquid–liquid phase separation (LLPS) and increased viscosity in highly concentrated solutions [ 9 – 12 ]. The LLPS of mAbs could result in a poor appearance and inconsistent vial-to-vial concentrations [ 9 ].…”

Section: Introductionmentioning

confidence: 99%

“…One of the key risks is the loss of colloidal stability, specifically undesirable liquid–liquid phase separation (LLPS) and increased viscosity in highly concentrated solutions [ 9 – 12 ]. The LLPS of mAbs could result in a poor appearance and inconsistent vial-to-vial concentrations [ 9 ].…”

Section: Introductionmentioning

confidence: 99%

Mitigation of liquid–liquid phase separation of a monoclonal antibody by mutations of negative charges on the Fab surface

et al. 2020

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Some monoclonal antibodies undergo liquid–liquid phase separation owing to self-attractive associations involving electrostatic and other soft interactions, thereby rendering monoclonal antibodies unsuitable as therapeutics. To mitigate the phase separation, formulation optimization is often performed. However, this is sometimes unsuccessful because of the limited time for the development of therapeutic antibodies. Thus, protein mutations with appropriate design are required. In this report, we describe a case study involving the design of mutants of negatively charged surface residues to reduce liquid–liquid phase separation propensity. Physicochemical analysis of the resulting mutants demonstrated the mutual correlation between the sign of second virial coefficient B 2 , the Fab dipole moment, and the reduction of liquid–liquid phase separation propensity. Moreover, both the magnitude and direction of the dipole moment appeared to be essential for liquid–liquid phase separation propensity, where electrostatic interaction was the dominant mechanism. These findings could contribute to a better design of mutants with reduced liquid–liquid phase separation propensity and improved drug-like biophysical properties.

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“…Physicalc hemistry offersa large toolboxo fs ophisticated experimentala nd theoretical methods that allow quantitativea nalysiso fm olecular-level details of solvation and crowding phenomena that help not only to better understand cellular processes, but they can also help in the development of diagnostic tools and therapeutic strategies of human diseases. [25,26] In particular,w ef ocus on the cosolvente ffects on the conformational equilibrium and kinetics of proteins and nucleic acids as well as enzymatic reactions, including their effectso nt he temperature and pressure dependence of these processes. We show how such effectsa re experimentally unveiled andc orrectly described in thermodynamic terms, and present afew representativee xamples.…”

Section: Introductionmentioning

confidence: 99%

Crowders and Cosolvents—Major Contributors to the Cellular Milieu and Efficient Means to Counteract Environmental Stresses

et al. 2017

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The free energy and conformational landscape of biomolecular systems as well as biochemical reactions depend not only on temperature and pressure, but also on the particular solution conditions. Such conditions include the effects of cosolvents (for example osmolytes) and macromolecular crowding, which are crucial components to understand the energetics and kinetics of biological processes in living system. Such conditions are also important for the understanding of many debilitating diseases, such as those where misfolding and amyloid formation of proteins are involved. Moreover, understanding their effects on biomolecular processes is prerequisite for designing industrially relevant enzymatic reactions, which seldom take place under neat conditions. Here, we review and discuss experimental and theoretical studies on the characterization of cosolvent and crowding induced effects in biologically relevant systems, approaching even the complexity of living organisms. In particular, we focus on cosolvent and crowding effects on the conformational equilibrium and folding kinetics of proteins and nucleic acids as well as on enzymatic reactions, including their effects on the temperature and pressure dependence of these processes. By presenting a few representative examples, we show how such effects are unveiled and described in thermodynamic and kinetic terms.

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Liquid-liquid phase separation of a monoclonal antibody at low ionic strength: Influence of anion charge and concentration

Cited by 46 publications

References 89 publications

Stress Factors in mAb Drug Substance Production Processes: Critical Assessment of Impact on Product Quality and Control Strategy

Stress Factors in mAb Drug Substance Production Processes: Critical Assessment of Impact on Product Quality and Control Strategy

Mitigation of liquid–liquid phase separation of a monoclonal antibody by mutations of negative charges on the Fab surface

Crowders and Cosolvents—Major Contributors to the Cellular Milieu and Efficient Means to Counteract Environmental Stresses

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