(b) DNA size distribution profile of a fraction with an amplification ratio of 40%. Conclusion We have evaluated the impact of process parameters (pH and conductivity), DNA size distribution and competition by HCP on DNA clearance capability and binding behavior on AEX chromatography media in the flowthrough mode. characterized based on a size exclusion qPCR assay. Results showed preferential binding of larger DNA fragments ( 409 base pairs). ? 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers is the concentration and is the volume of CHMFL-ABL-121 the feed weight or product collected, as noted. DNA breakthrough was not observed for the SuperQ runs for either the LS or the HS conditions (Table 2). Therefore, an additional run with DNA spike of 12,000 ng DNA/mg of mAb was conducted to see if DNA breakthrough at higher DNA weight would be observed. No breakthrough was seen at this very high spike level either. Results show that SuperQ resin provides 6.9 LRV of DNA clearance for a load challenge up to 2.3 mg of DNA/mL column volume. This binding capacity exceeded that of both membranes for the same weight pH and conductivity. Figures ?Figures44 and ?and55 show the DNA breakthrough curves for DNA load challenge up to 7 mg DNA/mL of membrane volume for Sartobind Q and Mustang Q, respectively. For Sartobind Q, DNA started to breakthrough at approximately 0. 8 mg DNA/mL membrane volume for both low and high DNA spike runs. Both runs experienced similar DNA profiles and overlaid when plotted against the amount of DNA loaded. This result suggests that the binding capacity of DNA is usually independent of CHMFL-ABL-121 the DNA weight concentration for the range studied. Data suggest a clearance capability of approximately 6 LRV, up to 1 1 mg DNA loaded/mL of membrane. Open in a separate window Physique 4 DNA breakthrough and DNA clearance as log10 reduction value for Sartobind Q at pH 6.0 and 15 mS/cm conductivity. Solid diamonds (?) and solid triangles () indicate DNA concentrations ( em C /em ) in the flowthrough fractions relative to initial DNA concentration in the load ( em C /em 0) for low and high DNA spike ratio, respectively. Open circles () and open triangles () indicate log DNA clearance for low spike and high spike ratio, respectively. Open in a separate window Physique 5 DNA breakthrough and DNA clearance as log10 reduction value for Mustang Q at pH 6.0 and 15 mS/cm conductivity. Solid circles (?) and solid triangles () indicate DNA concentrations in flowthrough fractions ( em C /em ) relative to initial DNA concentration in the load ( em C /em 0) for low and high DNA spike ratio, respectively. Open circles () and open triangles () indicate log DNA clearance for low spike and high spike ratio, respectively. Comparable DNA profiles were also seen in Mustang Q, although the breakthrough was observed CHMFL-ABL-121 at higher DNA weight (around 1.5 mg DNA/mL membrane volume). This difference in DNA binding capacity could be attributed to larger pore size of the Sartobind Q membrane which translates to less available surface area for binding.9 LRV greater than 5 can be obtained if DNA weight is limited to less than 0.8 and 1.5 mg/mL, respectively, for Sartobind Q and Mustang Q. Table 3 summarizes the DNA clearance capability for the AEX chromatography media evaluated. At the least favorable conditions for DNA binding, SuperQ resin resulted in the highest binding capacity for DNA and provided a higher LRV compared to both membranes. Assuming 100 ng DNA/mg mAb in the cell\free culture supernatant and a maximum mAb weight onto the AEX chromatography of 200 mg/mL resin, the maximum DNA weight is usually 20 g DNA/mL of column. The binding capacity of DNA is usually greater than 2.3 mg DNA/mL of column, which provides a safety factor for the SuperQ resin of more than 6.9 LRV of DNA. This analysis gives confidence around the AEX chromatography capability to obvious DNA, Rabbit Polyclonal to EDG2 even at the least favorable conditions for DNA binding. As a polishing step, the AEX chromatography is usually preceded by a capture step that by itself can typically provide a minimum of 1C3 LRV, providing a redundant step for DNA removal. Table 3 DNA Clearance Capability at pH 6.0 and 15 mS/cm Conductivity thead valign=”bottom” th align=”left” valign=”bottom” rowspan=”1″ colspan=”1″ Media /th th align=”center” valign=”bottom” rowspan=”1″ colspan=”1″ Capacity Before DNA Breakthrough (mg DNA/mL CV or MV) /th th align=”center” valign=”bottom” rowspan=”1″ colspan=”1″ DNA Clearance (log10) /th /thead SuperQ 2.3 6.9Mustang Q1.56.0Sartobind Q0.85.9 Open in a separate window CV?=?column volume; MV?=?membrane.
(b) DNA size distribution profile of a fraction with an amplification ratio of 40%