Purification of monoclonal antibodies from clarified cell culture fluid using Protein A capture continuous countercurrent tangential chromatography.

Recent studies using simple model systems have demonstrated that continuous countercurrent tangential chromatography (CCTC) has the potential to overcome many of the limitations of conventional Protein A chromatography using packed columns. The objective of this work was to optimize and implement a CCTC system for monoclonal antibody purification from clarified Chinese Hamster Ovary (CHO) cell culture fluid using a commercial Protein A resin. Several improvements were introduced to the previous CCTC system including the use of retentate pumps to maintain stable resin concentrations in the flowing slurry, the elimination of a slurry holding tank to improve productivity, and the introduction of an "after binder" to the binding step to increase antibody recovery. A kinetic binding model was developed to estimate the required residence times in the multi-stage binding step to optimize yield and productivity. Data were obtained by purifying two commercial antibodies from two different manufactures, one with low titer (∼ 0.67 g/L) and one with high titer (∼ 6.9 g/L), demonstrating the versatility of the CCTC system. Host cell protein removal, antibody yields and purities were similar to those obtained with conventional column chromatography; however, the CCTC system showed much higher productivity. These results clearly demonstrate the capabilities of continuous countercurrent tangential chromatography for the commercial purification of monoclonal antibody products.

Removal of lipopolysaccharides from protein-lipopolysaccharide complexes by nonflammable solvents.

During the recovery of recombinant proteins from gram negative bacteria, many of the methods used to extract proteins from cells release lipopolysaccharides (LPS, endotoxin) along with the protein of interest. In many instances, LPS will co-purify with the target protein due to specific or non-specific protein-LPS interactions. We have investigated the ability of alkanediols to effect the separation of LPS from protein-LPS complexes while the complexes are immobilized on ion exchange chromatographic resins. Proteins were complexed with fluorescently labeled LPS and bound to ion exchange resin. Alkanediol washes of the resins were preformed and the proteins eluted. Column eluates were monitored for LPS and protein by fluorescence and UV spectroscopy, respectively. Alkanediols were effective agents for dissociating LPS from protein-LPS complexes. The efficiency of LPS removal increased with increasing alkanediol chain length. The 1,2-alkanediol isomers were more effective than terminal alkanediol isomers in the separation of LPS from protein-LPS complexes, while the separation of LPS from protein-LPS complexes was more efficient on cation exchangers than on anion exchangers. In addition, it was noted during these investigations that the 1,2-alkanediols increased the retention time of the proteins on the ion exchange resins. Alkanediols provide a safer alternative to the use of other organics such as alcohols or acetonitrile for the separation of LPS from protein due to their lower toxicity and decreased inflammability. In addition, they are less costly than many of the detergents that have been used for similar purposes.

Ion chromatographic quantification of cyanate in urea solutions: estimation of the efficiency of cyanate scavengers for use in recombinant protein manufacturing.

The chaotrope urea is commonly used during recombinant protein manufacturing as a denaturant/solublizing agent. The adventitious accumulation of cyanate in urea solutions during product manufacturing can cause unwanted carbamylation of proteins, leading to alterations in drug product structure, stability and function. We have developed an ion chromatographic method to quantify cyanate production in urea solutions, suitable for analysis of samples from manufacturing process buffers. We discuss assay development, system suitability criteria and limitations on assay applicability. The assay has a linear range from 2 to 250 microM, with LOQ/LOD values of 6 and 2 microM, respectively. Assay accuracy through spike/recovery testing were established and both precision and intermediate precision were estimated. We assessed the utility of the assay by testing a variety of biological buffers and potential cyanate scavengers, which could be used during protein purification processes, for their ability to control the level of cyanate in 8 M urea solutions buffered over the range of pH 5-10. Our results demonstrate pH dependence for prevention of cyanate accumulation by these buffers/scavengers and indicate useful buffers, pH ranges, and additives for controlling cyanate accumulation during recombinant protein manufacturing. The pertinence of these approaches in preventing protein carbamylation during manufacturing are discussed.