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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

SEC chromatograms for IgG4 incubated under a range of low pH conditions. Top: pH 2.78 in solution. Middle: pH 3.05 after elution from column loaded with 12 mg IgG. Bottom: pH 2.92 after elution from column loaded with 25 mg IgG. Legends show incubation times to the nearest minute.
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fig0010: SEC chromatograms for IgG4 incubated under a range of low pH conditions. Top: pH 2.78 in solution. Middle: pH 3.05 after elution from column loaded with 12 mg IgG. Bottom: pH 2.92 after elution from column loaded with 25 mg IgG. Legends show incubation times to the nearest minute.

Mentions: Size exclusion chromatography was used to quantify IgG monomer and high molecular weight (HMW) species in the test samples. Monomer loss over time was chosen as the basis for quantification of aggregation rates. Under all sufficiently harsh conditions tested, monomers were partially converted to a range of high molecular weight species over time as can be seen in Fig. 2. A few major observations were made. Firstly, regardless of (perceived) harshness of conditions, with sufficient time, incubation at low pH resulted in an increasing front shoulder to the monomer peak and a small shift of the whole peak in the direction of shorter elution time. This is indicative of a monomer either with increased molecular weight, or with altered physical character that could accelerate its transit through the SEC column [5]. Harsher conditions and/or longer incubation times instigated these changes in the monomer peak. The pronounced front shoulder on the monomer peak (Fig. 2, top) may represent an individual population of monomer in a molten globule state [27]. Alternatively, it may be due to an oxidised species such as the oxidised tryptophan IgG2 monomer “pre-peak” species characterised by Wong et al. [28]. Another observation was that decrease in monomer peak area was proportional to increase in HMW species peak area, up to a point. After this point, (see top and middle plots in Fig. 2), the peak area for HMW species did not increase and the monomer front shoulder developed further; in the bottom plot (Fig. 2) the monomer peak continued to decline without either significant development of the monomer front shoulder or an increase in HMW species. These results indicate that aggregates whose molecular weights exceed the exclusion limit of the SEC column may form to varying extents depending on the conditions. Such species may have been retained inside the column or HPLC system, or been removed by centrifugation prior to sample injection onto the column.


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)

SEC chromatograms for IgG4 incubated under a range of low pH conditions. Top: pH 2.78 in solution. Middle: pH 3.05 after elution from column loaded with 12 mg IgG. Bottom: pH 2.92 after elution from column loaded with 25 mg IgG. Legends show incubation times to the nearest minute.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig0010: SEC chromatograms for IgG4 incubated under a range of low pH conditions. Top: pH 2.78 in solution. Middle: pH 3.05 after elution from column loaded with 12 mg IgG. Bottom: pH 2.92 after elution from column loaded with 25 mg IgG. Legends show incubation times to the nearest minute.
Mentions: Size exclusion chromatography was used to quantify IgG monomer and high molecular weight (HMW) species in the test samples. Monomer loss over time was chosen as the basis for quantification of aggregation rates. Under all sufficiently harsh conditions tested, monomers were partially converted to a range of high molecular weight species over time as can be seen in Fig. 2. A few major observations were made. Firstly, regardless of (perceived) harshness of conditions, with sufficient time, incubation at low pH resulted in an increasing front shoulder to the monomer peak and a small shift of the whole peak in the direction of shorter elution time. This is indicative of a monomer either with increased molecular weight, or with altered physical character that could accelerate its transit through the SEC column [5]. Harsher conditions and/or longer incubation times instigated these changes in the monomer peak. The pronounced front shoulder on the monomer peak (Fig. 2, top) may represent an individual population of monomer in a molten globule state [27]. Alternatively, it may be due to an oxidised species such as the oxidised tryptophan IgG2 monomer “pre-peak” species characterised by Wong et al. [28]. Another observation was that decrease in monomer peak area was proportional to increase in HMW species peak area, up to a point. After this point, (see top and middle plots in Fig. 2), the peak area for HMW species did not increase and the monomer front shoulder developed further; in the bottom plot (Fig. 2) the monomer peak continued to decline without either significant development of the monomer front shoulder or an increase in HMW species. These results indicate that aggregates whose molecular weights exceed the exclusion limit of the SEC column may form to varying extents depending on the conditions. Such species may have been retained inside the column or HPLC system, or been removed by centrifugation prior to sample injection onto the column.

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