Regeneration Studies of Anion-Exchange Chromatography Resins

LEVEL: INTERMEDIATE C hromatography columns can become contaminated by a variety of protein and nonprotein species during a purification campaign. Consequences of column contamination include an increase in backpressure, loss of signal resolution, altered product yield, and medium discoloration. Common chromatographic contaminants include • residual proteins • nucleic acids • lipids • endotoxins • viruses and bacteria • metal ions. Generally, methods for cleaning-inplace (CIP) and sanitization-in-place (SIP) of chromatographic resins are selected based on the interplay and relevance of three factors: ease of operation, historical experience, and performance requirements. In most cases, a column decontamination method chosen by a laboratory forms the basis not only for process validation, but also for subsequent scale-up. For this reason, an ideal scenario would be development and use of a generic decontamination method. At present, however, even taking into account the varying types of resins available, considerable disparity of CIP and SIP procedures is apparent in instructions available from manufacturers of chromatography resins. Many traditional cleaning solutions are used for CIP and SIP. Table 1 lists target contaminants of these solutions. Unquestionably, the cleaning strategy that has attracted the most attention and produced the most dependable results is the combination of sodium chloride (NaCl) and sodium hydroxide (NaOH). It has repeatedly proven effective in chromatography column decontamination. The key advantage of NaOH is its bactericidal action: NaOH inhibits the growth of and kills many bacteria and microorganisms. When NaOH–NaCl is applied to base-resistant chromatography resins supplied by various manufacturers, it has proven to be highly effective in validation studies for removal of residual proteins, viruses, and endotoxins (1, 2). In our investigation, we addressed the clearance of DNA because there is limited relevant information or supporting data on mass balance. DNA, being highly negatively charged, has strong affinity for the positively charged surfaces of anion-exchange resins. Any DNA not removed by a cleaning procedure will gradually accumulate over time and diminish column binding capacity and selectivity. Accordingly, we chose to examine chromatography issues such as postcleaning DNA recovery and selectivity, as are discussed here. We also touch upon simultaneous clearance of endotoxin and residual proteins.