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

Figure 7. HIC screening profiles on PPG at different monitor settings. Conditions as in Table 4. Red arrow indicates aggregates.
Most IgM monoclonals bind strongly to anion exchangers. Anion exchange capture is handicapped, however, because contaminating proteins consume a significant portion of the binding capacity.7 Stronger binding contaminants are an even greater liability for strong anion exchangers, such as Q, quaternary amino (QA), quaternary amino ethyl (QAE), and trimethylaminoethyl (TMAE), because they maintain sufficient positive charge in 1.0 M NaOH to retain DNA through multipoint binding of numerous phosphoryl residues. Combinations of 1–2 M NaCl with NaOH provide more effective cleaning than NaOH alone, but only column treatment with DNase has proven adequate to achieve quantitative removal.43,44 If capture by anion exchange is desired, it is prudent to use a weak anion exchanger, such as diethylaminoethyl (DEAE). The liability of binding competition by contaminating proteins is still present, but DEAE loses its charge in NaOH, thereby releasing DNA and other strongly bound contaminants.

Process Modeling

Figure 8. Preparative capture profile on cation exchange. Equilibrate column with 50 mM MES, pH 6.0. Load sample by inline dilution, 1 part CCS, 4 parts equilibration buffer. Wash with equilibration buffer. Wash with 10 mM sodium phosphate, pH 7.0. Elute with a 10 mM linear gradient to 250 mM sodium phosphate pH 7. Clean with 500 mM sodium phosphate (not shown).
After establishing a preliminary process order, loading conditions, dynamic binding capacity, and separation conditions at each step, it is useful to run the integrated process to provide a benchmark of overall process performance and economics. In-depth optimization of the individual steps before running the integrated process is premature. For example, one might expend considerable resources developing conditions to remove a difficult contaminant with one particular method, only to learn that it is removed effortlessly by another method. A good working process model highlights its own deficiencies, helps provide an order of priority for addressing them, and allows each to be evaluated in a meaningful context.

Figure 9. Reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of purification stages
Figure 8 provides an example of the sort of deficiency that benchmark evaluation can reveal. In this case, the wash and gradient conditions had been determined with small injections of CCS, and dynamic binding capacity had been estimated with hydroxyapatite-purified reference IgM. CCS loading volume was set at 80% of that capacity, with the expectation that all of the IgM would bind and elute solely in the gradient. No antibody was lost during sample application, but approximately 8% of the IgM eluted prematurely in the pH 7.0 wash. This fact indicated that the column was saturated, apparently due to competition from binding contaminants, and it suggested two possible corrections: reduce the sample volume, or reduce the wash and elution pH. Figure 9 illustrates product purity at various process stages.

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