With the worldwide appetite for biologics soaring—the U.S. alone accounts for roughly $100 billion annually, and that’s growing at 11% CAGR—efforts to optimize bioprocessing technology remain vigorous and varied.
Faster assays for cell-line screening, small-scale modeling to predict large-scale production effects, and new microfluidics able to finely control microenvironments are just a few of the many technologies on the horizon.
One of the most pressing concerns for all companies manufacturing biologics is preventing viral contamination; raw materials constitute a considerable portion of that risk. Switching to different raw materials to mitigate the risk is often done, particularly the migration away from materials of animal origin. The problem, however, is difficulty predicting the effects of these changes on legacy processes. Sofie Goetschalckx, Genzyme’s manufacturing cell culture science lead, technology division, discusses her firm’s practices.
“Reducing viral risk is our primary goal in changing materials at the moment, although sometimes we assess a second supplier so if one company goes out of business or can no longer provide the product, we will still have a supplier who can produce the material,” says Goetschalckx.
“We’ve developed qualified small-scale models. So we have a 10-liter cell culture model that is performing similar to our ‘at-scale’ 4,000 liter bioreactor, and we have a downstream model that performs similar to our overall product quality generated at scale,” she adds. “We take three different lots from the supplier so we have some variability in the raw material and run the small-scale models and see what the impact is.”
Building these small-scale models takes two to three years, according to Goetschalckx. One of the most problematic issues is controlling PCO2. “At small scale, PCO2 is completely different than at scale and has a huge impact on cell growth and recovered product quality. Our cells tend to grow a little better at scale than at small scale.” Genzyme adds more CO2 to the small-scale model process to compensate.
Sampling size also has an effect. At small scale the effect is much larger and more significant than at scale. “It turns out there are some differences that are challenging that you can solve but some you cannot solve,” she notes. Depending upon the results and the risk assessment on the criticality of the material, “we determine if we need additional data at scale in a kind of engineering run. Ultimately, this package of data will be reviewed by laboratory quality and also regulatory authorities to assess if you need to resubmit. Resubmission is very rare.”
Sialic Acid Content Analysis
Sialic acid (SA) is an important component for therapeutic proteins; it can prolong serum half-life, influence the biological activity, and improve solubility of proteins. Lam Raga Anggara Markley, Ph.D., scientist, Biogen Idec, discusses an improved high-throughput assay for SA content (high-throughput total sialic acid assay (HT-TSA)) that was developed at Biogen and based on his earlier work at MIT. One important application is faster screening of clones when thousands must be analyzed to winnow down possibilities for a particular bioprocess.
Several factors affect SA content analysis, among them intraclonal variability as well as production parameters and instability. Currently HPLC is most often used to measure SA content, but the process can take five days.
The new high-throughput method (HTM) is more accurate than the earlier version and “takes about 70 minutes, and we can analyze more than 100 samples in one day,” says Dr. Markley. Speed is the main advantage. “The downside is that HTM cannot distinguish Neu5Ac (N-Acetylneuraminic acid; NANA) from Neu5Gc (N-Glycolylneuraminic acid; NGNA) because they have the same fluorescence spectra. HPLC can distinguish the two.”
The assay method consists of four steps:
- Step 1. Crude culture samples are purified using a high-throughput protein purification that can simultaneously purify 96 samples within 30 minutes. This step renders the HTM specific to sialic acid bound to products of interest.
- Steps 2 & 3. The purified protein is denatured for 30 minutes using a surfactant in order to ensure complete cleavage of sialic acid by sialidase (EC 22.214.171.124) (five minutes) in the third step.
- Step 4. Sialic acid is converted into a fluorescent molecule by malononitrile derivatization (five minutes). The fluorescence intensity is used to calculate the sialic acid concentration, which is then divided by the purified protein concentration to obtain the sialic acid content.
“People may wonder when they should use HPLC and when to use the HTM assay. As a general rule, use HPLC when you don’t have a large number of samples and you need to know the level of the two acids. If you have hundreds of samples and need the data quickly, and don’t need to know both levels, you can use the high-throughput method,” Dr. Markley explains.