Highly Concentrated Monoclonal Antibody Solutions: Direct Analysis of Physical Structure and Thermal Stability

Harn, Nicholas R.; Allan, Christopher M.; Oliver, Cynthia N.; Middaugh, C. Russell

doi:10.1002/jps.20753

Cited by 143 publications

(113 citation statements)

References 32 publications

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“…This behavior suggests an amplification on the level of secondary structure before unfolding and has been observed for antibodies previously. 33,36 Above 70 C, the spectra decreased in intensity as the secondary structure was gradually lost (spectra omitted for clarity). Figure 2(b) shows near-UV spectra in the wavelength range from 250 to 350 nm recorded while ramping the sample temperature.…”

Section: Thermal Denaturationmentioning

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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Section: Thermal Denaturationmentioning

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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“…X-ray crystallography, nuclear magnetic resonance (NMR), circular dichroism (CD), and a number of spectroscopic techniques are routinely used for determination of secondary structure [19][20][21]. A recent study utilized deep ultraviolet resonance Raman (DUVRR) to sensitively characterize mAb secondary structure [22].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Ion Mobility-Mass Spectrometry for Comparative Analysis of Monoclonal Antibodies

Ferguson

Gucinski-Ruth

2016

J. Am. Soc. Mass Spectrom.

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Abstract. Analytical techniques capable of detecting changes in structure are necessary to monitor the quality of monoclonal antibody drug products. Ion mobility mass spectrometry offers an advanced mode of characterization of protein higher order structure. In this work, we evaluated the reproducibility of ion mobility mass spectrometry measurements and mobiligrams, as well as the suitability of this approach to differentiate between and/or characterize different monoclonal antibody drug products. Four mobiligram-derived metrics were identified to be reproducible across a multi-day window of analysis. These metrics were further applied to comparative studies of monoclonal antibody drug products representing different IgG subclasses, manufacturers, and lots. These comparisons resulted in some differences, based on the four metrics derived from ion mobility mass spectrometry mobiligrams. The use of collision-induced unfolding resulted in more observed differences. Use of summed charge state datasets and the analysis of metrics beyond drift time allowed for a more comprehensive comparative study between different monoclonal antibody drug products. Ion mobility mass spectrometry enabled detection of differences between monoclonal antibodies with the same target protein but different production techniques, as well as products with different targets. These differences were not always detectable by traditional collision cross section studies. Ion mobility mass spectrometry, and the added separation capability of collision-induced unfolding, was highly reproducible and remains a promising technique for advanced analytical characterization of protein therapeutics.

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“…8 The destabilization is probably because of the increased intermolecular contacts in the concentrated state and may be generally characteristic of proteins under such conditions. 9,10 It should be emphasized, however, that these conclusions were based on studies with only two mAbs whose behavior, in fact, differed significantly from one another.…”

Section: Introductionmentioning

confidence: 99%

Formulation Design and High-Throughput Excipient Selection Based on Structural Integrity and Conformational Stability of Dilute and Highly Concentrated IgG1 Monoclonal Antibody Solutions

Bhambhani

Kissmann

Joshi

et al. 2012

Journal of Pharmaceutical Sciences

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Highly Concentrated Monoclonal Antibody Solutions: Direct Analysis of Physical Structure and Thermal Stability

Cited by 143 publications

References 32 publications

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

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

Evaluation of Ion Mobility-Mass Spectrometry for Comparative Analysis of Monoclonal Antibodies

Formulation Design and High-Throughput Excipient Selection Based on Structural Integrity and Conformational Stability of Dilute and Highly Concentrated IgG1 Monoclonal Antibody Solutions

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