Shaken, Not Stirred: Mechanical Stress Testing of an IgG1 Antibody

Kiese, Sylvia; Papppenberger, Astrid; Frieß, Wolfgang; Mahler, Hanns‐Christian

doi:10.1002/jps.21328

Cited by 335 publications

(307 citation statements)

References 46 publications

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“…Whereas rigorous agitation should help in eliminating the least stable mAbs, it appears to be overly disruptive for differentiating more stable molecules. A similar concern was raised previously by Kiese et al 34 with respect to selecting the optimal type and duration of a mechanical stress. Results from a more recent systematic investigation by Eppler et al 14 demonstrate the possibility of fine tuning the agitation stress to produce practically relevant data.…”

Section: Agitation-induced Mab Aggregationmentioning

confidence: 55%

“…This also agrees with the fact that stirring can induce more aggregation than shaking, even in the complete absence of headspace. 34 It is worth mentioning, however, that aggregation in the presence of the interfacial area must be influenced by protein adsorption, which determines the surface coverage. It was shown previously that mAbs differ significantly in their surface affinity depending on overall hydrophobicity and pI.…”

Section: Agitation-induced Mab Aggregationmentioning

confidence: 99%

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The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

et al. 2010

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This study introduces a novel analytical approach for studying aggregation and phase separation of monoclonal antibodies (mAbs). The approach is based on using analytical scale cation-exchange chromatography (CEX) for measuring the loss of soluble monomer in the case of individual and mixed protein solutions. Native CEX outperforms traditional size-exclusion chromatography in separating complex protein mixtures, offering an easy way to assess mAb aggregation propensity. Different IgG1 and IgG2 molecules were tested individually and in mixtures consisting of up to four protein molecules. Antibody aggregation was induced by four different stress factors: high temperature, low pH, addition of fatty acids, and rigorous agitation. The extent of aggregation was determined from the amount of monomeric protein remaining in solution after stress. Consequently, it was possible to address the role of specific mAb regions in antibody aggregation by co-incubating Fab and Fc fragments with their respective full-length molecules. Our results revealed that the relative contribution of Fab and Fc regions in mAb aggregation is strongly dependent on pH and the stress factor applied. In addition, the CEX-based approach was used to study reversible protein precipitation due to phase separation, which demonstrated its use for a broader range of protein-protein association phenomena. In all cases, the role of Fab and Fc was clearly dissected, providing important information for engineering more stable mAb-based therapeutics.

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Section: Agitation-induced Mab Aggregationmentioning

confidence: 55%

Section: Agitation-induced Mab Aggregationmentioning

confidence: 99%

The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

et al. 2010

View full text Add to dashboard Cite

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“…These developing in silico approaches complement established accelerated stress studies that are performed in vitro to predict the shelf‐life and stability of biologics (Jain et al, 2017; Yang et al, 2013). Various methods are employed to generate such data including heating (Cheng et al, 2012; Hamrang et al, 2015), stirring (Luo et al, 2011; Sediq, Van Duijvenvoorde, Jiskoot, & Nejadnik, 2016), shaking (Kiese, Papppenberger, Friess, & Mahler, 2008; Rudiuk, Cohen‐Tannoudji, Huille, & Tribet, 2012), and simulation of transportation (Fleischman, Chung, Paul, & Lewus, 2017). The extent of aggregation, however, can be heavily dependent on the type of accelerated stress employed (Fleischman et al, 2017; Joubert, Luo, Nashed‐Samuel, Wypych, & Narhi, 2011; Tamizi & Jouyban, 2016).…”

Section: Introductionmentioning

confidence: 99%

Using extensional flow to reveal diverse aggregation landscapes for three IgG1 molecules

Willis

Kumar

Dobson

et al. 2018

Biotech & Bioengineering

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Monoclonal antibodies (mAbs) currently dominate the biopharmaceutical sector due to their potency and efficacy against a range of disease targets. These proteinaceous therapeutics are, however, susceptible to unfolding, mis‐folding, and aggregation by environmental perturbations. Aggregation thus poses an enormous challenge to biopharmaceutical development, production, formulation, and storage. Hydrodynamic forces have also been linked to aggregation, but the ability of different flow fields (e.g., shear and extensional flow) to trigger aggregation has remained unclear. To address this question, we previously developed a device that allows the degree of extensional flow to be controlled. Using this device we demonstrated that mAbs are particularly sensitive to the force exerted as a result of this flow‐field. Here, to investigate the utility of this device to bio‐process/biopharmaceutical development, we quantify the effects of the flow field and protein concentration on the aggregation of three mAbs. We show that the response surface of mAbs is distinct from that of bovine serum albumin (BSA) and also that mAbs of similar sequence display diverse sensitivity to hydrodynamic flow. Finally, we show that flow‐induced aggregation of each mAb is ameliorated by different buffers, opening up the possibility of using the device as a formulation tool. Perturbation of the native state by extensional flow may thus allow identification of aggregation‐resistant mAb candidates, their bio‐process parameters and formulation to be optimized earlier in the drug‐discovery pipeline using sub‐milligram quantities of material.

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“…15,16 In this study, we used the monoclonal IgG1 antibody Rituximab 17 as a model system to better understand the mechanisms leading to the formation of large aggregates when incubating a multidomain protein under weak thermal stress. Aggregation of antibodies due to various kinds of stress have been addressed in a number of studies, [18][19][20][21][22][23][24] with a few key observations highlighted later. In this context, we distinguish between the formation of oligomers (dimers, trimers, etc.)…”

Section: Introductionmentioning

confidence: 99%

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

et al. 2010

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Using an IgG1 antibody as a model system, we have studied the mechanisms by which multidomain proteins aggregate at physiological pH when incubated at temperatures just below their lowest thermal transition. In this temperature interval, only minor changes to the protein conformation are observed. Light scattering consistently showed two coupled phases: an initial fast phase followed by several hours of exponential growth of the scattered intensity. This is the exact opposite of the lagtime behavior typically observed in protein fibrillation. Dynamic light scattering showed the rapid formation of an aggregate species with a hydrodynamic radius of about 25 nm, which then increased in size throughout the experiment. Theoretical analysis of our light scattering data showed that the aggregate number density goes through a maximum in time providing compelling evidence for a coagulation mechanism in which aggregates fuse together. Both the analysis as well as size-exclusion chromatography of incubated samples showed the actual increase in aggregate mass to be linear and reach saturation long before all molecules had been converted to aggregates. The CH2 domain is the only domain partly unfolded in the temperature interval studied, suggesting a pivotal role of this least stable domain in the aggregation process. Our results show that for multidomain proteins at temperatures below their thermal denaturation, transient unfolding of a single domain can prime the molecule for aggregation, and that the formation of large aggregates is driven by coagulation.

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Shaken, Not Stirred: Mechanical Stress Testing of an IgG1 Antibody

Cited by 335 publications

References 46 publications

The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

Using extensional flow to reveal diverse aggregation landscapes for three IgG1 molecules

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

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