Figure 2
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After concentration and diafiltration, the purified MGG was pumped across a sterile filter (Opticap phillic filter with Durapore,
0.22-μm membrane) into a bag. A sample was drawn from the final purified MGG to determine the concentration and assess the
bioburden of the purified MGG. The purity of the polyclonal MGG was evaluated by running polyacrylamide gel electrophoresis,
followed by estimation of purity using QuantiScan software (Figure 2). The bioburden of the purified MGG was determined by
plating a sample on growth media and checking for microbial growth. The yield was calculated based on 3% scale results.
Results
Figure 2 shows the purification profile of the polyclonal MGG from mouse serum using the integrated disposable assembly. Comparison
of the integrated assembly purified MGG (well 10) to the control reveals that the purity in well 10 was very high. One band
each of the light chain and the heavy chain of the MGG were observed and contaminant bands seen in the control were absent.
Purity percentages were determined using QuantiScan Software.
Discussion
The proposed change from concentration/dialysis to ultrafiltration/diafiltration resulted in a significantly faster process
and reduced process time from two weeks to 25 hours thus improving turnaround times and productivity at the facility.
The changes to chromatographic buffer composition for both the equilibration buffer (from 10 mM PBS, pH 7.2, to 20 mM PBS,
pH 7.4) and elution buffer (from glycine HCL, 0.2 M, pH 2.8, to glycine HCL, 0.1 M, pH 2.8) had the following benefits: an
increase in final concentration to 79.28 mg/mL (minimum expected target concentration 25–50 mg/mL); 100% purity (minimum expected
target 95%); and an increase in yield of 96.64% (minimum expected target 90%). Reducing filter hold up losses by adopting
system rinse and air blow down may increase yields further.
The elimination of the azide also has potential for refinement caused by contamination issues with the dilution buffer. Future
work needs to be carried out in order to resolve the contamination issue in the dilution buffer. Using the purification process
described, the overall cost of operation for a 200-L batch was estimated to be $41.13/g of MGG.
Gazala Khan-Koticha, PhD, is a consultant for business and knowledge development, Lisa Nakakihara, PhD, is a senior manager for manufacturing operations, Lee Tucker is a scientific manager, John Scarcella is a research scientist, and Eric Rudolph, PhD, is a director for strategy and corporate development, all at Millipore Corporation, Billerica, MA, 781.533.2233, gazala_khan-koticha@millipore.com
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