Forced Degradation of Therapeutic Proteins

Hawe, Andrea; Wiggenhorn, Michael; Weert, Marco van de; Garbe, Joerg H.O.; Mahler, Hanns‐Christian; Jiskoot, Wim

doi:10.1002/jps.22812

Cited by 219 publications

(156 citation statements)

References 158 publications

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“…1) have been shown to represent the unfolding of either the F ab or the C H 3 domain depending on the antibody and solution conditions. 42,43 In addition, the T onset values, marking the temperature at which the first structural transition initiates (an important value for formulation stability considerations), 44 were also determined. A summary of the 3 T m and T onset values are listed in Table 1.…”

Section: Resultsmentioning

confidence: 99%

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

Majumdar

Esfandiary²,

Bishop³

et al. 2015

mAbs

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(2015) Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life, mAbs, 7:1, 84-95, DOI: 10.4161/19420862.2014.985494 To link to this article: https://doi.org/10. 4161/19420862.2014 This study compares the local conformational dynamics and physical stability of an IgG1 mAb (mAb-A) with its corresponding YTE (M255Y/S257T/T259E) mutant (mAb-E), which was engineered for extended half-life in vivo. Structural dynamics was measured using hydrogen/deuterium (H/D) exchange mass spectrometry while protein stability was measured with differential scanning calorimetry (DSC) and size exclusion chromatography (SEC). The YTE mutation induced differences in H/D exchange kinetics at both pH 6.0 and 7.4. Segments covering the YTE mutation sites and the FcRn binding epitopes showed either subtle or no observable differences in local flexibility. Surprisingly, several adjacent segments in the C H 2 and distant segments in the V H , C H 1, and V L domains had significantly increased flexibility in the YTE mutant. Most notable among the observed differences is increased flexibility of the 244-254 segment of the C H 2 domain, where increased flexibility has been shown previously to correlate with decreased conformational stability and increased aggregation propensity in other IgG1 mAbs (e.g., presence of destabilizing additives as well as upon de-glycosylation or methionine oxidation). DSC analysis showed decreases in both thermal onset (T onset ) and unfolding (T m 1) temperatures of 7 C and 6.7 C, respectively, for the C H 2 domain of the YTE mutant. In addition, mAb-E aggregated faster than mAb-A under accelerated stability conditions as measured by SEC analysis. Hence, the relatively lower physical stability of the YTE mutant correlates with increased local flexibility of the 244-254 segment, providing a site-directed mutant example that this segment of the C H 2 domain is an aggregation hot spot in IgG1 mAbs.

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Section: Resultsmentioning

confidence: 99%

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

Majumdar

Esfandiary²,

Bishop³

et al. 2015

mAbs

View full text Add to dashboard Cite

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“…Numerous studies investigated the effect of these stress conditions on the formation of aggregates in biopharmaceutical formulations. [3][4][5][6][7] Despite extensive efforts to minimize negative effects on the molecules, 8,9 the formation of undesired high molecular weight species (HMWs) and aggregates cannot be avoided completely. Protein aggregation is therefore an important quality attribute that may have an effect on potency and pharmacokinetics.…”

Section: Introductionmentioning

confidence: 99%

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

Plath

Ringler

Graff-Meyer

et al. 2016

mAbs

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(2016) Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs, mAbs, 8:5, 928-940, DOI: 10.1080/19420862.2016 To link to this article: https://doi.org/10. 1080/19420862.2016 ABSTRACTThe formation of undesired high molecular weight species such as dimers is an important quality attribute for therapeutic monoclonal antibody formulations. Therefore, the thorough understanding of mAb dimerization and the detailed characterization mAb dimers is of great interest for future pharmaceutical development of therapeutic antibodies. In this work, we focused on the analyses of different mAb dimers regarding size, surface properties, chemical identity, overall structure and localization of possible dimerization sites. Dimer fractions of different mAbs were isolated to a satisfactory purity from bulk material and revealed 2 predominant overall structures, namely elongated and compact dimer forms. The elongated dimers displayed one dimerization site involving the tip of the Fab domain. Depending on the stress applied, these elongated dimers are connected either covalently or non-covalently. In contrast, the compact dimers exhibited non-covalent association. Several interaction points were detected for the compact dimers involving the hinge region or the base of the Fab domain. These results indicate that mAb dimer fractions are rather complex and may contain more than one kind of dimer. Nevertheless, the overall appearance of mAb dimers suggests the existence of 2 predominant dimeric structures, elongated and compact, which are commonly present in preparations of therapeutic mAbs.

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“…Protein aggregates and particles are important quality attributes of therapeutic protein formulations (1)(2)(3). Especially micron-sized aggregates (subvisible protein particles) (4) are considered as critical due to their potential risk of enhancing an immunogenic response (5).…”

Section: Introductionmentioning

confidence: 99%

Flow Imaging Microscopy for Protein Particle Analysis—A Comparative Evaluation of Four Different Analytical Instruments

Zölls

Weinbuch

Wiggenhorn³

et al. 2013

AAPS J

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Abstract. Flow imaging microscopy was introduced as a technique for protein particle analysis a few years ago and has strongly gained in importance ever since. The aim of the present study was a comparative evaluation of four of the most relevant flow imaging microscopy systems for biopharmaceuticals on the market: Micro-Flow Imaging (MFI)4100, MFI5200, Flow Cytometer And Microscope (FlowCAM) VS1, and FlowCAM PV. Polystyrene standards, particles generated from therapeutic monoclonal antibodies, and silicone oil droplets were analyzed by all systems. The performance was critically assessed regarding quantification, characterization, image quality, differentiation of protein particles and silicone oil droplets, and handling of the systems. The FlowCAM systems, especially the FlowCAM VS1, showed high-resolution images. The FlowCAM PV system provided the most precise quantification of particles of therapeutic monoclonal antibodies, also under impaired optical conditions by an increased refractive index of the formulation. Furthermore, the most accurate differentiation of protein particles and silicone oil droplets could be achieved with this instrument. The MFI systems provided excellent size and count accuracy (evaluated with polystyrene standards) especially the MFI5200 system. This instrument also showed very good performance for protein particles, also in case of an increased refractive index of the formulation. Both MFI systems were easier to use and appeared more standardized regarding measurement and data analysis as compared to the FlowCAM systems. Our study shows that the selection of the appropriate flow imaging microscopy system depends strongly on the main output parameters of interest and it is recommended to decide based on the intended application.

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Forced Degradation of Therapeutic Proteins

Cited by 219 publications

References 158 publications

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

Flow Imaging Microscopy for Protein Particle Analysis—A Comparative Evaluation of Four Different Analytical Instruments

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