Improving Quality and Safety
Of the several significant microbial contaminants that plague mammalian cell culture manufacturing, bacteria are more common but viruses are more devastating. Adventitious viral infections—as opposed to viruses emerging from the genomes of cultured cells—always lead to loss of product and often to extended plant shutdowns.
No company is immune from viruses, no matter how rigorous their sourcing and cleaning practices. Genzyme, Genentech, and Amgen have all been affected, with the minute virus of mice (MVM) particularly troublesome.
“The only way to deal with a viral infection is to quarantine the entire building, throw away everything you possibly can, shrinkwrap the facility, and fumigate,” says Bob Weaver, senior scientist at Amgen.
Raw materials, including media ingredients, are a high-risk entry point for viral contamination. Today’s preference for better defined, animal component-free media requires 50 or more discrete media ingredients, each one multiplying the risk of MVM or other viruses entering the production cycle. “One virus particle can contaminate an entire plant,” Weaver observes.
The solution that Amgen has adopted is risk mitigation at point of entry. Ingredients are treated with one of two virucidal technologies borrowed from other industries: ultraviolet-C (UV-C) and high-temperature short-time pasteurization (HTST). Widely used in the sterilization of water, beverages, and blood plasma, short-wave UV-C includes the germicidal 253.7 nm wavelength. HTST is widely used in the food industry and is similar to other types of heat treatments used in biotech.
Genentech applies these treatments after formulation, which assures that the media is sterile at the point of use. According to Weaver every large mammalian cell culture facility is either using or considering deployment of media sterilization. “Although it’s expensive we’re considering it for all our products. If you’re in this business long enough, chances are that sooner or later your process will be exposed to some type of virus.”
Regulators have recently become concerned with media components that are not cleared by purification unit operations. The targets are not amino acids and nutrients, but additives such as anti-foaming agents and detergents. “These substances are not listed in the drug substance specification, as purification steps are expected to remove them,” notes Suzanne Aldington, Ph.D., a group leader at Lonza Biologics. But chromatography and membrane filtration do not always do the job adequately.
While these substances are not considered unsafe at the levels at which they are added, regulators are nevertheless concerned because their use and carry-over through downstream processing has not been subjected to a thorough risk-assessment study.
Chromatography, buffer exchange, membrane adsorption, and filtration are known to remove most of these materials but no-one knows to what extent they do. For example, detergents should wash through a protein A column, ultrafiltration membranes trap impurities below 10 kDa, and antifoaming agents stick to filters.
Still, Dr. Aldington’s group is looking into assays that will quantify additives at each purification step. These may include spiking studies where known quantities of impurity are loaded, at small scale, onto a column and the eluent tested by HPLC.
It is unknown whether FDA and its European counterpart, EMA, will require specific assays for common media and process additives, but Dr. Aldington suggests that the likelihood of having to add specific purification steps to clear trace additives is not very great.