The next step was to assess the effect of processing on the quality of medium.
This was done by HTST, treating larger, unspiked batches of 50% glucose solution at the pilot scale and comparing it with untreated batches. The HTST treatment was carried out in an Armfield FT74X UHT pilot-scale system, which uses a water-heated shell-in-tube heat exchanger, with the glucose solution heated to and held at 102°C for 10 seconds. Both the treated and untreated batches were sterile filtered, and then packaged into 10 L EVA bags.
The heating and processing profiles for both the pilot and benchtop scale were similar. The cooling time post-processing in the pilot-scale unit was significantly longer than in the benchtop system. As it remains at a higher temperature for longer, the potential for higher levels of viral inactivation may be achieved in this larger-scale system; the benchtop system represents a worst-case scenario.
Performance of both treated and untreated glucose solution in cell cultures was then evaluated. It was added at a concentration of 5.5 g/L to glucose-free, chemically defined CHO medium, and inoculated with SAFC’s CHOZN® GS–/– ZFN modified CHO cells at a density of 3.0 x 105 cells/mL. Cultures were counted daily to determine growth and viability.
The cultures were maintained in a Multitron incubator at 37°C and 5% CO2 until viabilities dropped below 70%. Spent media samples were collected on days 7, 10, 12, and 14 for analysis.
The growth curves for medium that contained glucose that was HTST-treated at pilot scale showed similar growth kinetic profiles to medium containing the nonthermally processed solution, with culture viabilities comparable across treated and nontreated media. HTST processing of the 50% glucose solution had no effect on the growth characteristics of the medium.
A diverse set of media and concentrates were also performance tested following HTST treatment at 102ºC for 10 seconds. Serum-free and chemically defined media, supplements such as vitamins, hydrolysate concentrates and amino acid concentrates were all included. In each case, no significant differences were observed in analytical data or cell culture performance using CHO cells following HTST treatment.
When introducing a new media or feed to a process involving HTST, or integrating HTST into an existing process, it is critical to test the compatibility of the media and supplements. Some components such as phosphates and high concentrations of divalent cations can precipitate at elevated temperatures, so verification at pilot scale is essential.
These results demonstrate that the small-scale, static, benchtop model, when used in concert with the pilot-scale HTST system, facilitates viral clearance evaluation and a performance assessment of complex media formulations or media components.