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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

Schematic of experimental design where incubation at low pH follows protein A chromatography (a). Incubation is assumed to begin when the elution peak begins, denoted t0. Fractions containing approximately the first two thirds of the elution peak were at pH conditions considerably higher than the elution buffer pH. IgG concentration and pH in fraction 3 were the conditions reproduced in solution experiments for comparability with column experiments. A precise representation of the experimental outcome can be seen in (b), a chromatogram from a protein A chromatography run. The solid line (left y-axis) represents the IgG elution peak and the dotted line (right y-axis) indicates the pH of column effluent, as measured by the in-line pH probe. Here, flow of elution buffer begins at 7 mL elution volume. The last two fractions of the elution peak, indicated within vertical lines in (b), were at pH conditions low enough to induce aggregation.
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fig0005: Schematic of experimental design where incubation at low pH follows protein A chromatography (a). Incubation is assumed to begin when the elution peak begins, denoted t0. Fractions containing approximately the first two thirds of the elution peak were at pH conditions considerably higher than the elution buffer pH. IgG concentration and pH in fraction 3 were the conditions reproduced in solution experiments for comparability with column experiments. A precise representation of the experimental outcome can be seen in (b), a chromatogram from a protein A chromatography run. The solid line (left y-axis) represents the IgG elution peak and the dotted line (right y-axis) indicates the pH of column effluent, as measured by the in-line pH probe. Here, flow of elution buffer begins at 7 mL elution volume. The last two fractions of the elution peak, indicated within vertical lines in (b), were at pH conditions low enough to induce aggregation.

Mentions: Initial experiments were done in solution under a range of low pH conditions and IgG4 concentrations in order to determine the basic behaviour of the mAb at low pH and identify a useful pH range to work in. Typical conditions and time-courses for protein A chromatography and virus inactivation at low pH were taken into account. Time-consumption and reproducibility of experiments was also considered. Additionally, the formulation buffer of the purified mAb contributed to experimental design decisions; as the formulation buffer already contained a high concentration of glycine (0.27 M), glycine–HCl buffers were used to lower the pH in solution experiments, and for product elution in column experiments. Glycine–HCl is often used as an affinity chromatography elution buffer, though citrate or acetate buffers are more common in bioprocessing when a slightly higher elution pH is required [23–26]. In the case of this work, using glycine–HCl reduced the number of sample manipulation steps and potential confounding effects this might have on aggregation data. The pH range chosen for experiments covers a larger range than would usually be considered for industrial purification [6,7,9,10,21], as it is required to plot correlations between aggregation rates and low pH. Column experiments were carried out as shown in Fig. 1. Incubation at the elution pH was assumed to begin at the first sign of the elution peak (increase in absorbance at 280 nm), denoted t0 in Fig. 1a. It is assumed that the mAb has been exposed to the elution buffer at this stage. The column effluent does not approach pH 3 until the tail end of the peak, i.e. Fraction 3, but marking t0 earlier eliminates the possibility of exaggerating the column effect on aggregation rate. The IgG4 concentration in Fraction 3 (Fig. 1b) was within the range tested in solution, supporting comparability between in-solution and on-column data.


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)

Schematic of experimental design where incubation at low pH follows protein A chromatography (a). Incubation is assumed to begin when the elution peak begins, denoted t0. Fractions containing approximately the first two thirds of the elution peak were at pH conditions considerably higher than the elution buffer pH. IgG concentration and pH in fraction 3 were the conditions reproduced in solution experiments for comparability with column experiments. A precise representation of the experimental outcome can be seen in (b), a chromatogram from a protein A chromatography run. The solid line (left y-axis) represents the IgG elution peak and the dotted line (right y-axis) indicates the pH of column effluent, as measured by the in-line pH probe. Here, flow of elution buffer begins at 7 mL elution volume. The last two fractions of the elution peak, indicated within vertical lines in (b), were at pH conditions low enough to induce aggregation.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig0005: Schematic of experimental design where incubation at low pH follows protein A chromatography (a). Incubation is assumed to begin when the elution peak begins, denoted t0. Fractions containing approximately the first two thirds of the elution peak were at pH conditions considerably higher than the elution buffer pH. IgG concentration and pH in fraction 3 were the conditions reproduced in solution experiments for comparability with column experiments. A precise representation of the experimental outcome can be seen in (b), a chromatogram from a protein A chromatography run. The solid line (left y-axis) represents the IgG elution peak and the dotted line (right y-axis) indicates the pH of column effluent, as measured by the in-line pH probe. Here, flow of elution buffer begins at 7 mL elution volume. The last two fractions of the elution peak, indicated within vertical lines in (b), were at pH conditions low enough to induce aggregation.
Mentions: Initial experiments were done in solution under a range of low pH conditions and IgG4 concentrations in order to determine the basic behaviour of the mAb at low pH and identify a useful pH range to work in. Typical conditions and time-courses for protein A chromatography and virus inactivation at low pH were taken into account. Time-consumption and reproducibility of experiments was also considered. Additionally, the formulation buffer of the purified mAb contributed to experimental design decisions; as the formulation buffer already contained a high concentration of glycine (0.27 M), glycine–HCl buffers were used to lower the pH in solution experiments, and for product elution in column experiments. Glycine–HCl is often used as an affinity chromatography elution buffer, though citrate or acetate buffers are more common in bioprocessing when a slightly higher elution pH is required [23–26]. In the case of this work, using glycine–HCl reduced the number of sample manipulation steps and potential confounding effects this might have on aggregation data. The pH range chosen for experiments covers a larger range than would usually be considered for industrial purification [6,7,9,10,21], as it is required to plot correlations between aggregation rates and low pH. Column experiments were carried out as shown in Fig. 1. Incubation at the elution pH was assumed to begin at the first sign of the elution peak (increase in absorbance at 280 nm), denoted t0 in Fig. 1a. It is assumed that the mAb has been exposed to the elution buffer at this stage. The column effluent does not approach pH 3 until the tail end of the peak, i.e. Fraction 3, but marking t0 earlier eliminates the possibility of exaggerating the column effect on aggregation rate. The IgG4 concentration in Fraction 3 (Fig. 1b) was within the range tested in solution, supporting comparability between in-solution and on-column data.

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