Purification of IgM Monoclonal Antibodies - Manufacturing challenges surround the use of IgM monoclonal - BioPharm International


Purification of IgM Monoclonal Antibodies
Manufacturing challenges surround the use of IgM monoclonal

BioPharm International Supplements

Aggregate Removal

SEC supports effective aggregate removal for IgMs, but its low productivity is unattractive for manufacturing operations.30,31 The primary handicap is the sample volume, typically in the range of 2–5% Cv. This limitation is compounded by flow rates potentially as low as 20 cm/hr.7 A theoretical benefit of SEC is that it simultaneously equilibrates the sample for the next step, but this benefit is moot for most IgMs because equilibrating a sample to ion exchange conditions on an SEC column is likely to result in formation of turbidity and to jeopardize product quality. It may result in the IgM precipitating on the column. This is the basis of an IgM purification technique called euglobulin partitioning chromatography, in which the antibody is retained by SEC media at conductivity values too low to support solubility.7,32–34 Soluble contaminants flow through on sample application. The IgM is subsequently eluted by increasing the salt concentration.

Many high-capacity alternatives are available for aggregate removal. Although it is impossible to predict which will best serve a particular IgM, it is likely that the screening conditions suggested above will reveal one or more methods that offer useful levels of aggregate reduction. Hydroxyapatite has frequently proven effective for removal of aggregates and polymers from IgG, IgA, and IgM monoclonal antibodies.16–18,35–37 HIC and ion exchange frequently provide worthy aggregate fractionation of IgG aggregates, and can be reasonably expected to do so with IgMs as well. Figure 7 illustrates promising separation of IgM aggregates on a PPG HIC column.

If the native abilities of the methods in a process are not sufficient to achieve adequate aggregate reduction, it may be possible to enhance them. Recent investigations have demonstrated the ability of 3.75–7.5% polyethylene glycol (PEG) to promote effective IgG aggregate removal on ion exchangers and hydroxyapatite, even when no separation is apparent in the absence of PEG.38 Larger proteins are more responsive than IgG to the effects of PEG, suggesting that this treatment should be even more effective for IgMs.39 PEG is economical, is protein-stabilizing, and is an approved inactive ingredient in a large number of parenteral formulations.40,41

Process Sequencing

The combination of good capacity, excellent contaminant removal, minimal sample preparation, and consistent applicability for most IgMs makes hydroxyapatite a good default candidate for capture. Typically, the eluting salt concentration is sufficiently low, with dilution, to make ion exchange practical as a second step. The high salt tolerance of HIC makes it a good candidate for a second step, but HIC is poorly suited to capture because of the large amounts of salt that are required. This would be an impediment even if the salt could be added directly to the sample, but the usual need for inline dilution multiplies salt requirements to a prohibitive level. Assuming that a concentration of 1.2 M ammonium sulfate was required for binding, and that this was achieved by inline dilution of 1 part sample with 4 parts 1.5 M ammonium sulfate, 0.99 kg ammonium sulfate would be required for every liter of CCS. Column loading time would be an issue, since the dilution factor would quintuple sample volume.

The strong binding of the IgM shown in Figure 4 suggests the feasibility of cation exchange capture. Published data, however, indicate that cation exchange retention is highly variable for IgMs, so it should not be relied on as a default method.7 Under the best of circumstances, mild reduction of pH and 3–5 fold dilution of the CCS will probably be necessary to obtain good binding capacity. Another positive feature of cation exchange capture is that it removes CCS contaminants that can affect hydroxyapatite, principally including metal ions and chelators. Iron binds to and discolors hydroxyapatite, but published studies indicate that separation performance is unaffected.42 Continuous presence of at least 5 mM phosphate and a minimum pH of 6.5 generally stabilizes hydroxyapatite, but how well it does so in the presence of chelators in CCS remains to be evaluated. One important restriction with cation exchange as a capture method is that it cannot use citrate buffers if hydroxyapatite is the next step: citrate is a calcium chelator.

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