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Improve Recovery and Reproducibility with Pre-Treatment of Analytical TSK-GEL G3000SWXL Size Exclusion Columns

cular weight species during SEC analysis of the post-protein A fraction was highly variable, signaling possible adsorption of the sample to the packing materials. A pre-treatment experimental protocol with BSA was administered to reduce the variability.


Conditions of SEC analysis:
Column: TSKgel G3000SWXL, 30cm x 7.8mm, 5 micron

Elution: Phosphate Buffered Saline, pH 6.5

Sample: 50 mL injection volume, 50 mg load

Detection: 280 nm

Instrument: Waters 2690 Alliance


Results:
As can be seen in Figure 1 , recovery of the high molecular weight region, expressed as a percent of total area recovery (% HMW), of the protein A purified sample was 1.1%. Upon injecting 10 aliquots x 20 mL of 5 mg/mL bovine serum albumin (BSA) onto the column, the recovery of the %HMW species increased to 3.5%. In the absence of BSA treatment, protein A purified samples historically returned %HMW values of 1.0% to 1.4% when a column was new and gradually increased to 3.5% - 4.0% after as many as 60 injections. Interestingly, this result was only apparent with the protein A purified sample. Table 1 shows that BSA pre-treatment had no effect on the samples obtained from the downstream chromatography steps. Further characterization work indicated that elevated levels of host cell proteins (HCP) were present in the protein A purified samples with respect to the post polishing samples. The HCP were found to elute in the HMW region of the analytical SEC profile. Furthermore, when the protein A purified samp
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