Understanding the relationship between biotherapeutic protein stability and solid-liquid interfacial shear in constant region mutants of IgG1 and IgG4.
Bottom Line: Five variants of IgG1 and IgG4 antibodies were created using combinations of two discrete triple amino acid sequence mutations denoted TM and YTE.Molecular modeling shows the modifications' effects on the antibody structures and indicates a possible role for Fc conformation and Fab-Fc docking in determining suspended protein stability.The data introduce the PDC value as an orthogonal stability indicator, complementary to traditional thermal methods, allowing lead antibody selection based on a more full understanding of process stability.
Affiliation: The Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK; MedImmune, Granta Park, Cambridge, CB21 6GH, UK.Show MeSH
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Mentions: The candidates for stability studies were produced from the IgG1 and IgG4 formats of an antibody. These formats were modified using combinations of two discrete, defined triple amino acid sequence mutations, TM39 and YTE,40 to give five variants with known pharmacological properties (Fig. 2). The five different antibodies were processed in the shear device at 6000, 7500, 9000, and 12,000 rpm. The antibody concentration selected was 1 mg/mL based on previous work and experimental evidence showing little change in the protein decay coefficient (PDC) between 0.5 and 10 mg/mL and a desire to minimize sample consumption. Each processed sample was cloudy when removed from the chamber, indicating the presence of aggregate. The insoluble material was removed by centrifugation prior to SE-HPLC. The area under the monomer curve at 15 min intervals was fitted to an exponential decay curve:where C(t) is the monomer concentration (mg/mL) at time t (h), C0 is the monomer concentration at time zero and k is the PDC (h−1). Statistical t-tests performed on the groups of results from each shear rate gave a confidence level of 97.5% that the trend seen was significant. The monomer reduction profiles for IgG1 TM YTE (Fig. 3) are shown as an example of the profiles seen. Greater monomer loss occurred at increased disk speeds indicating that the stability of the antibody was affected by processing at a range of disk speeds in the device.
Affiliation: The Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK; MedImmune, Granta Park, Cambridge, CB21 6GH, UK.