Larry J. Cummings is a consulting scientist involved in antibody purification at Bio-Rad Laboratories. “Bio-Rad has collaborated with the pharma industry, going back to 1981, working with ascites fluid. As the technology has evolved over the years, the favored strategy for antibody purification is to do a capture step with protein A, followed by an ion-exchange step and the final purification on our turnkey product, CHT™ Ceramic Hydroxyapatite media.”
According to Cummings, protein A has long been favored for antibody capture, given its abilities to bind immunoglobulins from cell culture harvests; reduce host-cell proteins, nucleic acids, endotoxins, and viruses; as well as its flexibility over a wide range of conditions. However, the elution conditions may promote aggregate formation, and a major shortcoming is its tendency to leach into the product.
“Protein A developed a high visibility in the industry because it provided high affinity and selectivity, while capturing a moderate load of antibody,” Cummings continues. “Very few materials had that capability.”
However, the demands of higher antibody-expression levels coming from the pharma industry, combined with the shortcomings of protein A, have driven companies to search for more productive alternatives. A number of companies, including EMD Chemicals, Tosoh Biosciences, Bio-Rad, and Life Technologies, have developed high-capacity cation-exchange resins. Given the long-term standing of protein A in antibody processing, these relatively new products will take some time to be accepted by the industry.
Cummings argues that his company’s product, Nuvia™ S, offers a number of important features, principally its performance as a high-capacity cation-exchange medium, providing antibody binding of 100 to 200 mg/mL.
In Cummings’ view, Nuvia S is a good candidate for antibody capture from cell culture harvests, as well as for intermediate purification steps. Although most of the DNA, endotoxins, and viruses flow through unbound, residual impurities including host-cell proteins must be removed following capture. Given that the cleaning conditions for the Nuvia S product are much less harsh than those required for protein A, concerns over antibody leaching and aggregation are eliminated.
“Moreover, antibody aggregates can be separated from monomeric antibody at loads up to 100 mg/mL, with a high recovery of the monomer,” he advised. “Fragments of light chain have been separated during antibody capture, using an intermediate wash step prior to the elution of the intact antibody.”
Additionally, industry practice is to conduct a viral inactivation step and then remove the inactivated virus, residual host cell protein and DNA with an anion-exchange resin. This step is given over to the Nuvia Q, which has a dynamic capacity for proteins of about 200 mg/mL. The Nuvia Q ligand, whose functionality is a quartenary ammonium group, is recognized for its viral and DNA reduction properties. The base chemistry is a proprietary polymer, consisting of rigid particles with large pores to accommodate strong flow properties and fast mass transit.
The final polishing step removes residual aggregates with CHT Ceramic Hydroxyapatite in the cation-exchange mode. While the complete platform is designed for whole IgG antibody purification, Cummings indicates that it could also be used for both minibody and antibody fragment-purification protocols.