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Protein A chromatography increases monoclonal antibody aggregation rate during subsequent low pH virus inactivation hold.

Mazzer AR, Perraud X, Halley J, O'Hara J, Bracewell DG - J Chromatogr A (2015)

Bottom Line: Yet, a more limited set of evidence suggests that low pH may not be the sole cause of aggregation in protein A chromatography, rather, other facets of the process may contribute significantly.Similar experiments were implemented in the absence of a chromatography step, i.e. IgG4 aggregation at low pH.Rate constants for aggregation after protein A chromatography were considerably higher than those from low pH exposure alone; a distinct shift in aggregation rates was apparent across the pH range tested.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Engineering, University College London, Bernard Katz Building, Gordon Street, London WC1H 0AH, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

Rates of monomer loss at 2.7 mg/mL IgG under different pH conditions in solution only (filled symbols and solid lines) and after elution from a protein A chromatography column (open symbols, dashed lines and dotted lines) (a–c). Different pH conditions are shown in the plot legends. Curves for column runs across the full pH range are shown together in (d). Error bars show the standard deviation for each time point based on full experimental repeats, n = 2. All data sets were fitted with exponential decay curves as was done for initial solution only data. All fits were significant with adjusted r2 > 0.98 and P < 0.01.
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fig0025: Rates of monomer loss at 2.7 mg/mL IgG under different pH conditions in solution only (filled symbols and solid lines) and after elution from a protein A chromatography column (open symbols, dashed lines and dotted lines) (a–c). Different pH conditions are shown in the plot legends. Curves for column runs across the full pH range are shown together in (d). Error bars show the standard deviation for each time point based on full experimental repeats, n = 2. All data sets were fitted with exponential decay curves as was done for initial solution only data. All fits were significant with adjusted r2 > 0.98 and P < 0.01.

Mentions: In Fig. 5a–c monomer loss over time under various pH conditions is plotted for column and solution experiments side by side. All column experiment data was fitted with exponential decay curves as was done for solution data. Fig. 5d shows all column experiment curves side by side on a single unbroken scale. Examining the plots in Fig. 5, the initial rate of monomer consumption and the position and behaviour of the plateau can be compared for the different test conditions. Due to the sharp drop-off in pH sensitivity between pH 2.7 and pH 3.0, the data in Fig. 5a–c is displayed in three narrow ranges: pH 2.78–2.86 (a), pH 2.90–2.95 (b) and pH 3.03–3.11 (c). Note that the x-axis scales differ for each plot and also contain scale breaks. The pH values indicate the pH at which the IgG was incubated after elution from the column, which was the same as the pH of the original elution buffer. In all cases, inclusion of the protein A chromatography step immediately before incubation resulted in a faster rate of monomer decay at any given pH. Exponential decay kinetics to a point of apparent equilibrium was maintained in column experiments as in solution. This suggests that the chromatography step does not significantly change the basic aggregation mechanism, but does accelerate it.


Protein A chromatography increases monoclonal antibody aggregation rate during subsequent low pH virus inactivation hold.

Mazzer AR, Perraud X, Halley J, O'Hara J, Bracewell DG - J Chromatogr A (2015)

Rates of monomer loss at 2.7 mg/mL IgG under different pH conditions in solution only (filled symbols and solid lines) and after elution from a protein A chromatography column (open symbols, dashed lines and dotted lines) (a–c). Different pH conditions are shown in the plot legends. Curves for column runs across the full pH range are shown together in (d). Error bars show the standard deviation for each time point based on full experimental repeats, n = 2. All data sets were fitted with exponential decay curves as was done for initial solution only data. All fits were significant with adjusted r2 > 0.98 and P < 0.01.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4582070&req=5

fig0025: Rates of monomer loss at 2.7 mg/mL IgG under different pH conditions in solution only (filled symbols and solid lines) and after elution from a protein A chromatography column (open symbols, dashed lines and dotted lines) (a–c). Different pH conditions are shown in the plot legends. Curves for column runs across the full pH range are shown together in (d). Error bars show the standard deviation for each time point based on full experimental repeats, n = 2. All data sets were fitted with exponential decay curves as was done for initial solution only data. All fits were significant with adjusted r2 > 0.98 and P < 0.01.
Mentions: In Fig. 5a–c monomer loss over time under various pH conditions is plotted for column and solution experiments side by side. All column experiment data was fitted with exponential decay curves as was done for solution data. Fig. 5d shows all column experiment curves side by side on a single unbroken scale. Examining the plots in Fig. 5, the initial rate of monomer consumption and the position and behaviour of the plateau can be compared for the different test conditions. Due to the sharp drop-off in pH sensitivity between pH 2.7 and pH 3.0, the data in Fig. 5a–c is displayed in three narrow ranges: pH 2.78–2.86 (a), pH 2.90–2.95 (b) and pH 3.03–3.11 (c). Note that the x-axis scales differ for each plot and also contain scale breaks. The pH values indicate the pH at which the IgG was incubated after elution from the column, which was the same as the pH of the original elution buffer. In all cases, inclusion of the protein A chromatography step immediately before incubation resulted in a faster rate of monomer decay at any given pH. Exponential decay kinetics to a point of apparent equilibrium was maintained in column experiments as in solution. This suggests that the chromatography step does not significantly change the basic aggregation mechanism, but does accelerate it.

Bottom Line: Yet, a more limited set of evidence suggests that low pH may not be the sole cause of aggregation in protein A chromatography, rather, other facets of the process may contribute significantly.Similar experiments were implemented in the absence of a chromatography step, i.e. IgG4 aggregation at low pH.Rate constants for aggregation after protein A chromatography were considerably higher than those from low pH exposure alone; a distinct shift in aggregation rates was apparent across the pH range tested.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Engineering, University College London, Bernard Katz Building, Gordon Street, London WC1H 0AH, United Kingdom.

No MeSH data available.


Related in: MedlinePlus