Reversible NaCl-induced aggregation of a monoclonal antibody at low pH: Characterization of aggregates and factors affecting aggregation

Bickel, Fabian; Herold, Eva Maria; Signes, Alba; Romeijn, Stefan; Jiskoot, Wim; Kiefer, H.

doi:10.1016/j.ejpb.2016.07.020

Cited by 45 publications

(37 citation statements)

References 46 publications

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“…To our knowledge, this is the first study that systematically controlled both pH and ionic strength during low pH incubation as well as after neutralization. Overall, our observations are in line with literature reports for other mAbs under comparable conditions (Bickel et al, ; Jin et al, ; Mazzer, Perraud, Halley, O'Hara, & Bracewell, ; Skamris et al, ). Therefore, we believe that our conclusions will hold for the majority of mAb molecules.…”

Section: Discussionsupporting

confidence: 93%

Understanding mAb aggregation during low pH viral inactivation and subsequent neutralization

Wälchli

Ressurreição

Vogg

et al. 2019

Biotech & Bioengineering

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Monoclonal antibodies (mAbs) and related recombinant proteins continue to gain importance in the treatment of a great variety of diseases. Despite significant advances, their manufacturing can still present challenges owing to their molecular complexity and stringent regulations with respect to product purity, stability, safety, and so forth. In this context, protein aggregates are of particular concern due to their immunogenic potential. During manufacturing, mAbs routinely undergo acidic treatment to inactivate viral contamination, which can lead to their aggregation and thereby to product loss. To better understand the underlying mechanism so as to propose strategies to mitigate the issue, we systematically investigated the denaturation and aggregation of two mAbs at low pH as well as after neutralization. We observed that at low pH and low ionic strength, mAb surface hydrophobicity increased whereas molecular size remained constant. After neutralization of acidic mAb solutions, the fraction of monomeric mAb started to decrease accompanied by an increase on average mAb size. This indicates that electrostatic repulsion prevents denatured mAb molecules from aggregation under acidic pH and low ionic strength, whereas neutralization reduces this repulsion and coagulation initiates. Limiting denaturation at low pH by D-sorbitol addition or temperature reduction effectively improved monomer recovery after neutralization. Our findings might be used to develop innovative viral inactivation procedures during mAb manufacturing that result in higher product yields. K E Y W O R D SANS fluorescence, downstream processing, monoclonal antibodies, protein aggregation, protein unfolding, viral inactivation

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Section: Discussionsupporting

confidence: 93%

Understanding mAb aggregation during low pH viral inactivation and subsequent neutralization

Wälchli

Ressurreição

Vogg

et al. 2019

Biotech & Bioengineering

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“…122 The effect of salts depends on their nature and concentration and may be divided between salting-in (stabilization) and salting-out (precipitation), high salt concentration (high ionic strength) being more in favor of aggregation salting-out. 123,124 The unchanged secondary structure analyses were however in favor of the retention of a native-like secondary structure even in the precipitated state, and mAb aggregation may be at least partially reversible by salt dilution. The type of salt also greatly influences the aggregation kinetics, with Na þ being the ion causing the smallest increase in aggregation formation (when compared to Ca 2þ or K þ , at pH 4), but in certain cases, the addition of the correct anion (sulfate) restored an IgG 2 to native configuration, at pH 3.…”

Section: Excipientsmentioning

confidence: 98%

Physicochemical Stability of Monoclonal Antibodies: A Review

Basle

Chennell

Tokhadzé

et al. 2020

Journal of Pharmaceutical Sciences

280

162

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Monoclonal antibodies (mAbs) are subject to instability issues linked to their protein nature. In this work, we review the different mechanisms that can be linked to monoclonal antibodies instability, the parameters, and conditions affecting their stability (protein structure and concentration, temperature, interfaces, light exposure, excipients and contaminants, and agitation) and the different analytical methods used for appropriate physicochemical stability studies: physical stability assays (aggregation, fragmentation, and primary, secondary, and tertiary structure analysis), chemical stability assays and quantitative assays. Finally, data from different published stability studies of mAbs formulations, either in their reconstituted form, or in diluted ready to administer solutions, was compiled. Overall, the physicochemical stability of mAbs is linked to numerous factors such as formulation, environment, and manipulations, and must be thoroughly investigated using several complementary analytical techniques, each of which allowing specific characterization information to be harvested. Several stability studies have been published, some of them showing possibilities of extended stability. However, those data should be questioned due to potential lacks in study methodology.

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“…[11][12][13] The protein is exposed to various buffer and salt solutions necessitated by the chromatographic and filtration steps of the process, which can also induce some aggregate formation. [14][15][16][17] Many of the process steps involve filtration, a process that creates a complex set of stress conditions including shear effects, cavitation, localized high concentrations of protein, and interactions at the filter membrane surface. This can even result in the formation of a gel layer 18 which is then sloughed out into the bulk as aggregates and can also act as a nucleus for further aggregate growth.…”

Section: Process Impurities and Microbiological Contaminantsmentioning

confidence: 99%

“…Furthermore, protein-protein interactions can also lead to reversible association of mAbs, typically under conditions at which a repulsive net charge is not dominating the overall interactions, observed at relatively net neutral charge, 88 or when charges are screened due to high ionic strength. 15 Breaking repulsive proteinprotein interactions typically reduce the solubility of mAbs. Nevertheless, depending on temperature, the type of salt (Hofmeister series), salt concentration, and protonation state of the protein (net positive charge at pH < pI; net neutral charge at pHp I; net negative charge at pH > pI), complex scenarios of solubility reduction and solubility enhancement ("salting in") have been reported.…”

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

confidence: 99%

“…The low pH hold study should also cover a range of reasonable low pH conditions that are potentially predictive of the behavior at the chosen pH for viral inactivation. 2 Owing to drastic changes known to occur in protein folding 153 and other 15,84,121,137,150,151 physicochemical properties for most proteins, choice of extreme pH conditions (such as pH 2) may not be meaningful to assess true risk of instability in the viral inactivation solvent condition. Finally, better methods to introduce an acid neutralization agent needs to be developed to reduce the impact of sudden exposure to high pH and ionic strength.…”

Section: Risk Mitigation For Exposure To Ph Extremesmentioning

confidence: 99%

See 1 more Smart Citation

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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Reversible NaCl-induced aggregation of a monoclonal antibody at low pH: Characterization of aggregates and factors affecting aggregation

Cited by 45 publications

References 46 publications

Understanding mAb aggregation during low pH viral inactivation and subsequent neutralization

Understanding mAb aggregation during low pH viral inactivation and subsequent neutralization

Physicochemical Stability of Monoclonal Antibodies: A Review

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

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