Limits...
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

Semi-log plot of monomer decay rate, R0, against pH for solution-only experiments (filled triangles) and experiments including a protein A chromatography step (open circles). For column experiments, the typical concentration of the incubated elution fraction was 2.7 mg/mL. Solution-only data seen in Fig. 4 was pooled due to overlapping 95% confidence intervals for the linear trends. For solution-only data points, x error bars represent the measurement error of the laboratory pH probe/metre, as reported by the manufacturer. For chromatography experiments, x error bars correspond to measured error based on three replicate measurements (with a micro pH probe). For both solution-only and column experiments, y error bars represent the standard error for the R0 parameter (Eq. (2)) obtained from least squares fitting. A linear trend was fitted to each data set, as shown by solid lines; dashed/dotted lines represent 95% confidence limits for each fit. Both fits were significant with P < 0.01 and adjusted r2 of 0.97 and 0.90 for solution-only and column experiments, respectively.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4582070&req=5

fig0035: Semi-log plot of monomer decay rate, R0, against pH for solution-only experiments (filled triangles) and experiments including a protein A chromatography step (open circles). For column experiments, the typical concentration of the incubated elution fraction was 2.7 mg/mL. Solution-only data seen in Fig. 4 was pooled due to overlapping 95% confidence intervals for the linear trends. For solution-only data points, x error bars represent the measurement error of the laboratory pH probe/metre, as reported by the manufacturer. For chromatography experiments, x error bars correspond to measured error based on three replicate measurements (with a micro pH probe). For both solution-only and column experiments, y error bars represent the standard error for the R0 parameter (Eq. (2)) obtained from least squares fitting. A linear trend was fitted to each data set, as shown by solid lines; dashed/dotted lines represent 95% confidence limits for each fit. Both fits were significant with P < 0.01 and adjusted r2 of 0.97 and 0.90 for solution-only and column experiments, respectively.

Mentions: Fig. 7 illustrates how the protein A chromatography step causes a shift in the rate of monomer loss compared to low pH incubation alone. It is clear that two separate trends exist for R0 against pH for solution-only and on-column experiments. In Fig. 7, 95% confidence bands were calculated using the standard error for the R0 parameter (obtained from least squares fitting) for weighting. The linear fit lines for column and solution-only R0 values are almost parallel; this supports the theory that the aggregation mechanism is accelerated, but not significantly altered, by the chromatography step. The data represents the minimum difference between after-column and solution-only rates, in that the earliest reasonable start time for low pH incubation was used for column experiments (see Section 3.1).


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)

Semi-log plot of monomer decay rate, R0, against pH for solution-only experiments (filled triangles) and experiments including a protein A chromatography step (open circles). For column experiments, the typical concentration of the incubated elution fraction was 2.7 mg/mL. Solution-only data seen in Fig. 4 was pooled due to overlapping 95% confidence intervals for the linear trends. For solution-only data points, x error bars represent the measurement error of the laboratory pH probe/metre, as reported by the manufacturer. For chromatography experiments, x error bars correspond to measured error based on three replicate measurements (with a micro pH probe). For both solution-only and column experiments, y error bars represent the standard error for the R0 parameter (Eq. (2)) obtained from least squares fitting. A linear trend was fitted to each data set, as shown by solid lines; dashed/dotted lines represent 95% confidence limits for each fit. Both fits were significant with P < 0.01 and adjusted r2 of 0.97 and 0.90 for solution-only and column experiments, respectively.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig0035: Semi-log plot of monomer decay rate, R0, against pH for solution-only experiments (filled triangles) and experiments including a protein A chromatography step (open circles). For column experiments, the typical concentration of the incubated elution fraction was 2.7 mg/mL. Solution-only data seen in Fig. 4 was pooled due to overlapping 95% confidence intervals for the linear trends. For solution-only data points, x error bars represent the measurement error of the laboratory pH probe/metre, as reported by the manufacturer. For chromatography experiments, x error bars correspond to measured error based on three replicate measurements (with a micro pH probe). For both solution-only and column experiments, y error bars represent the standard error for the R0 parameter (Eq. (2)) obtained from least squares fitting. A linear trend was fitted to each data set, as shown by solid lines; dashed/dotted lines represent 95% confidence limits for each fit. Both fits were significant with P < 0.01 and adjusted r2 of 0.97 and 0.90 for solution-only and column experiments, respectively.
Mentions: Fig. 7 illustrates how the protein A chromatography step causes a shift in the rate of monomer loss compared to low pH incubation alone. It is clear that two separate trends exist for R0 against pH for solution-only and on-column experiments. In Fig. 7, 95% confidence bands were calculated using the standard error for the R0 parameter (obtained from least squares fitting) for weighting. The linear fit lines for column and solution-only R0 values are almost parallel; this supports the theory that the aggregation mechanism is accelerated, but not significantly altered, by the chromatography step. The data represents the minimum difference between after-column and solution-only rates, in that the earliest reasonable start time for low pH incubation was used for column experiments (see Section 3.1).

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