Prediction of Key Analytes in Mammalian Cell Culture Media with On-Line near Infrared Spectroscopy

Introduction B iopharmaceuticals are therapeutic, large molecules such as proteins (e.g. antibodies) derived from biological sources that emulate natural human response to disease. Antibodies can be produced by bioengineered microbial cells but they have limited direct application in human therapy. Bioengineers make use of mammalian cells because they can produce a better match of the natural human antibodies and are not rejected as foreign. Antibodies for human therapy are generally produced in bioreactors using Chinese hamster ovary (CHO) cells. Monoclonal antibodies, produced by specifi c CHO cell lines, have been widely used and approved by the FDA for numerous human therapies ranging from auto immune disease to cancer treatment. This has led to a multi-billion dollar industry worldwide. Bioprocess applications have taken advantage of near infrared (NIR) spectroscopy for the prediction of analytes in growth media since the early 1990s. NIR has great potential for in-line monitoring of bioprocess media. Product titre (antibody) yield can be enhanced by predicting key analytes directly in a bioreactor in real-time allowing feedback for control and optimisation of nutrient levels as well as checking ammonia concentration. Daily off-line wet chemistry testing can introduce microbial or viral contamination. If analytes are sampled periodically throughout the day, the results are often not available for hours or even days after sampling or can be of questionable accuracy. In contrast, NIR technology can use transmission probes installed directly into a bioreactor to analyse feed stock and product accurately and in real-time. NIR analysis can monitor metabolic parameters in culture media allowing a high level of control and thereby increasing cell life and titre production. NIR spectra can be used effectively to predict many fermentation parameters simultaneously once the calibration models are developed. Experimental A System II Foss NIRSystems Process Analytics process spectrophotometer with a 3 m fi bre (210 illumination fi bres and 210 collection fi bres) and an Ingold fi bre-optic probe with a 4 mm fi xed pathlength was installed into a 100 L bioreactor in a pilot plant. An Ingold transfl ectance probe similar to the one seen in Figure 1 was used for this experiment with the exception that it had a fi xed pathlength of 4 mm (rather than an adjustable pathlength as shown). The probe provided high absorbance of low level analytes while maintaining high throughput within the 0.0–6.0 AU dynamic range of the indium gallium arsenide (InGaAs) detector. Since the probe can withstand 300°C temperature and 5000 psi pressure, it was steam sterilised in the bioreactor. An internal reference fi bre in the NIR process analyser allows continuous updating of the reference throughout the 12 day culture run, thus facilitating compensation for any variation in background spectral characteristics during the process run. Each spectrum consists of 32 reference scans and 32 sample scans with absorbance data being collected at 2 nm intervals over the spectral range of 800–2100 nm. Scans were collected at the rate of one per hour throughout the 12 day culture cycle. (If more continuous spectral information were required, NIR spectra could be collected continuously and with up to nine bioreactors multiplexed in series.) Off-line sampling was done once per day to obtain reference values by which the regression models were created using the Vision chemometric software that comes with the instrument. After the bioreactor with the inserted NIR probe was sterilised-in-place (SIP), the fed-batch culture of mammalian (CHO) cells was started and NIR spectra were collected and archived for 12 days.