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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

IgG monomer loss over time in solution at (a) 0.9 mg/mL, (b) 2.7 mg/mL and (c) 4.5 mg/mL. Different symbols represent different incubation pH conditions: circles, pH 3.03; open squares, pH 2.95; filled squares, pH 2.78. Error bars show the standard deviation for each point based on full experimental repeats, n = 2. Exponential decay curves were fitted to the data using the equation y = y0 + AeR0.x (see Section 3.2 for equation specifics). In (a), for pH 2.78 the last time point (2.18 h) was excluded from the curve fit (see Section 3.3.1). All fits were significant with adjusted r2 > 0.99 and P < 0.01.
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fig0015: IgG monomer loss over time in solution at (a) 0.9 mg/mL, (b) 2.7 mg/mL and (c) 4.5 mg/mL. Different symbols represent different incubation pH conditions: circles, pH 3.03; open squares, pH 2.95; filled squares, pH 2.78. Error bars show the standard deviation for each point based on full experimental repeats, n = 2. Exponential decay curves were fitted to the data using the equation y = y0 + AeR0.x (see Section 3.2 for equation specifics). In (a), for pH 2.78 the last time point (2.18 h) was excluded from the curve fit (see Section 3.3.1). All fits were significant with adjusted r2 > 0.99 and P < 0.01.

Mentions: The IgG4 was found to be highly sensitive to pH within a critical range. At pH conditions lower than pH 2.8 aggregation was rapid; plateau occurred in less than 30 min. However, at pH > 3.0 completion took approximately 4 h. It should be noted that the plateau was not found at the point of complete monomer loss, and varied depending on pH conditions; lower pH conditions resulted in greater total monomer loss (Fig. 3). It is possible that what appears as a plateau in monomer depletion actually represents a point of reversible equilibrium between species at one or more stages of the aggregation process. An ancillary experiment was carried out in which monomer purified from initial aggregation runs was re-exposed to low pH under the same conditions as in the initial runs. Recovered monomer was found to display near-identical aggregation behaviour to initial monomer (data not shown). This indicates that the surviving monomer population is not distinct from the aggregating portion, supporting the possibility of an equilibrium mechanism. Despite the fairly large IgG concentration range tested (five-fold), concentration appeared to have little effect on aggregation kinetics, and observed differences in plateau did not follow a clear trend. Thus we hypothesise that aggregation plateau was determined by pH-dependent equilibrium between unfolded and native or re-folded monomers. While this could provide an interesting topic for more detailed investigation, it will not be the focus of this study. Instead, we concentrate primarily on initial aggregation rates as well as apparent total monomer loss.


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)

IgG monomer loss over time in solution at (a) 0.9 mg/mL, (b) 2.7 mg/mL and (c) 4.5 mg/mL. Different symbols represent different incubation pH conditions: circles, pH 3.03; open squares, pH 2.95; filled squares, pH 2.78. Error bars show the standard deviation for each point based on full experimental repeats, n = 2. Exponential decay curves were fitted to the data using the equation y = y0 + AeR0.x (see Section 3.2 for equation specifics). In (a), for pH 2.78 the last time point (2.18 h) was excluded from the curve fit (see Section 3.3.1). All fits were significant with adjusted r2 > 0.99 and P < 0.01.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig0015: IgG monomer loss over time in solution at (a) 0.9 mg/mL, (b) 2.7 mg/mL and (c) 4.5 mg/mL. Different symbols represent different incubation pH conditions: circles, pH 3.03; open squares, pH 2.95; filled squares, pH 2.78. Error bars show the standard deviation for each point based on full experimental repeats, n = 2. Exponential decay curves were fitted to the data using the equation y = y0 + AeR0.x (see Section 3.2 for equation specifics). In (a), for pH 2.78 the last time point (2.18 h) was excluded from the curve fit (see Section 3.3.1). All fits were significant with adjusted r2 > 0.99 and P < 0.01.
Mentions: The IgG4 was found to be highly sensitive to pH within a critical range. At pH conditions lower than pH 2.8 aggregation was rapid; plateau occurred in less than 30 min. However, at pH > 3.0 completion took approximately 4 h. It should be noted that the plateau was not found at the point of complete monomer loss, and varied depending on pH conditions; lower pH conditions resulted in greater total monomer loss (Fig. 3). It is possible that what appears as a plateau in monomer depletion actually represents a point of reversible equilibrium between species at one or more stages of the aggregation process. An ancillary experiment was carried out in which monomer purified from initial aggregation runs was re-exposed to low pH under the same conditions as in the initial runs. Recovered monomer was found to display near-identical aggregation behaviour to initial monomer (data not shown). This indicates that the surviving monomer population is not distinct from the aggregating portion, supporting the possibility of an equilibrium mechanism. Despite the fairly large IgG concentration range tested (five-fold), concentration appeared to have little effect on aggregation kinetics, and observed differences in plateau did not follow a clear trend. Thus we hypothesise that aggregation plateau was determined by pH-dependent equilibrium between unfolded and native or re-folded monomers. While this could provide an interesting topic for more detailed investigation, it will not be the focus of this study. Instead, we concentrate primarily on initial aggregation rates as well as apparent total monomer loss.

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