Relating Protein–Protein Interactions and Aggregation Rates From Low to High Concentrations

Ghosh, Rabindra; Calero-Rubio, César; Saluja, Atul; Roberts, Christopher J.

doi:10.1016/j.xphs.2016.01.004

Cited by 70 publications

(122 citation statements)

References 49 publications

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“…These are formulated to minimize or avoid protein aggregation, high viscosity, phase separation, and opalescence among other potential stability and product quality issues. [4][5][6][7][8] Several solution conditions are adjusted to achieve a desirable formulation, including pH, buffer type and concentration, and salt/cosolute concentrations. Accounting for the range of potential formulations creates a combinatorically large set of possible conditions.…”

Section: Introductionmentioning

confidence: 99%

“…7 Net PPI depend on both the protein structure and the solution conditions (e.g., pH, total ionic strength [TIS], and excipients). 5,[10][11][12][13][14][15][16] PPI can be quantified experimentally with a number of techniques, including static light scattering (SLS), dynamic light scattering (DLS), analytical ultracentrifugation, self-interaction chromatography, and small-angle neutron or X-ray scattering. 5,7,8,15,[17][18][19] All of these techniques typically require measurements as a function of protein concentration and relevant solution conditions.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Electrostatically Mediated Protein-Protein Interactions for Monoclonal Antibodies: A Combined Experimental and Coarse-Grained Molecular Modeling Approach

Ferreira

Calero-Rubio

Sathish³

et al. 2019

Journal of Pharmaceutical Sciences

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111

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Electrostatically Mediated Protein-Protein Interactions for Monoclonal Antibodies: A Combined Experimental and Coarse-Grained Molecular Modeling Approach

Ferreira

Calero-Rubio

Sathish³

et al. 2019

Journal of Pharmaceutical Sciences

Self Cite

111

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“…Furthermore, the dimer levels approach an equilibrium following 60 days of incubation further supporting the reversibility of dimer. This type of concentration dependent self‐association has been shown for mAbs in general where increased concentration leads to increased self‐association of protein molecules …”

Section: Resultsmentioning

confidence: 58%

Effect of protein cryoconcentration and processing conditions on kinetics of dimer formation for a monoclonal antibody: A case study on bioprocessing

Mehta¹,

Subramanian²,

D'Mello³

et al. 2019

Biotechnology Progress

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Monoclonal antibodies (mAbs) may be prone to self‐association leading to formation of dimers, trimers, or other high molecular weight species during bio‐processing. In order to implement appropriate manufacturing control strategies during bio‐processing, it is important to understand various real life bio‐processing conditions where such self‐associations may manifest. One such case study is presented here of increase in dimer content for an mAb during scale‐up bio‐processing and the approach taken to understand the under‐lying mechanism. In this example, a therapeutic mAb demonstrated a consistently higher dimer values (~0.5% higher) in the drug product (DP) during release when compared to the same value measured in the corresponding drug substance (DS) lot. This observation was interesting since the DS was supplied frozen, and the DS and DP share the same formulation composition and therefore investigation of this dimer change was the scope of the characterization study. Variable path length spectroscopy and size exclusion chromatography was used for protein quantification and to monitor %dimer respectively during characterization of fill‐finish unit operations. At the start of DP manufacturing process, immediately after thaw of bulk DS, a protein concentration gradient was observed and the concentration ranged from 90 mg/mL (top of container) to 210 mg/mL (bottom of container). The dimerization kinetics in the same DS container was dependent on concentration with higher concentrations demonstrating higher rates of dimerization. After the bulk DS was mixed for further processing, %dimer in purified bulk DS was quantitated to be approximately 1.4% which is identical to levels observed during scale‐up manufacturing of DP. After each unit operation, the in‐process samples tested for %dimer showed a gradual increase in dimer as a function of time over the next 7 days accumulating to 1.8% dimer at the end of DP manufacturing process. Samples subjected to static incubation at 2–8°C and room temperature (RT; 15–25°C) showed a gradual increase in dimer over the same time frame; however, the rate of increase in dimer at RT was higher compared to samples stored at 2–8°C. The results from this demonstrate two important key findings: self‐association kinetics of mAbs could be exacerbated by protein cryoconcentration and temperature conditions during bioprocessing. Since these two parameters are commonly encountered during manufacturing, the proposed mitigation strategy is to ensure homogeneity of the bulk DP during processing. The temperature dependent self‐association kinetics of mAb could be mitigated by processing at lower temperature (e.g., 2–8°C) and by storing the finished DP at lower temperature after manufacturing. The results from this study also highlight the criticality of setting slightly wider specifications for DP compared to DS following ICH Q6B guidelines.

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“…Previous work within our group has looked at protein features related to protein solubility, in particular the lack of positively charged surface patches (Chan et al, 2013), the ratio of lysine to arginine residues (Warwicker et al, 2014), and the stability of individual Fab domains (Hebditch et al, 2017b). Experimental studies (Chari et al, 2009;Esfandiary et al, 2015;Neergaard et al, 2013;Yearley et al, 2013;Calero-Rubio et al, 2017;Roberts et al, 2014a;Ghosh et al, 2016;Inouye et al, 2016;Schermeyer et al, 2017), as well as computational approaches Lilyestrom et al, 2013;Corbett et al, 2017;Kuhn et al, 2017), have been aimed at understanding the solution behaviour of proteins and biologics.…”

Section: Introductionmentioning

confidence: 99%

Computational investigation of protein surface polarity and pH-dependence: application to antibodies shows that antibody-antigen interfaces tend to be relatively polar

Hebditch

Warwicker

2018

Preprint

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Protein instability leads to reversible self-association and irreversible aggregation which is a major concern for developing new biopharmaceutical leads. Protein solution behaviour is dictated by the physicochemical properties of the protein and the solution. Optimising protein solutions through experimental screens and targeted protein engineering can be a difficult and time consuming processes. Here, we describe further development of the protein-sol web tool, which was previously restricted to protein solubility prediction from amino acid sequence. Tools are presented for calculating and mapping patches of hydrophobicity and charge on the protein surface. In addition, predictions of folded state stability and net charge are displayed as a heatmap for a range of pH and ionic strength conditions. The tool is evaluated in the context of the interfaces present in Fab fragments and their antigens. Surprisingly, antibody-antigen interfaces are, on average, at least as polar as Fab surfaces. This benchmarking process provides the user with thresholds with which to assess non-polar surface patches, and possible solubility implications, in proteins of interest. Stability heatmaps compare favourably with experimental data for CH2 and CH3 domains. Display and quantification of surface polarity and pH / ionic strength dependence will be useful generally for investigation of protein biophysics.

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Relating Protein–Protein Interactions and Aggregation Rates From Low to High Concentrations

Cited by 70 publications

References 49 publications

Electrostatically Mediated Protein-Protein Interactions for Monoclonal Antibodies: A Combined Experimental and Coarse-Grained Molecular Modeling Approach

Electrostatically Mediated Protein-Protein Interactions for Monoclonal Antibodies: A Combined Experimental and Coarse-Grained Molecular Modeling Approach

Effect of protein cryoconcentration and processing conditions on kinetics of dimer formation for a monoclonal antibody: A case study on bioprocessing

Computational investigation of protein surface polarity and pH-dependence: application to antibodies shows that antibody-antigen interfaces tend to be relatively polar

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