Quimica

Near-Infrared Spectroscopy for Rapid, Simultaneous Monitoring
of Multiple Components in Mammalian Cell Culture
Cory Card, Brent Hunsaker, Tom Smith, and Jeffrey Hirsch
Reprinted with permission from BioProcess International 6(3): p 58-67 (March 2008)

he use of cellular physiology to Figure 1A: Concentrations of glucose and lactate throughout the course of the experiment make targetmolecules has been � practiced for centuries, with early examples being the production of ������� � wine and beer through yeast ������� fermentation. Single (e.g., bacteria and � yeast) and multicellular (plant or animal) organisms can be harnessed to produce � otherwise chemically complex, low-yield, or chemically uncharacterized materials. � These include “lock-and-key” receptor complexes with perfectstereochemical � specificity, large-scale protein scaffolds, or antibiotics. One example is penicillin, � with a sensitive β-lactam ring structure at its core (1). Mass-production of � penicillin began several years before its first synthesis was even published (2). � In the 20th century, designer �������� �������� �������� �������� ��������� ��������� biological synthesis entered the age of ����mass production. The first large-scale manufacture of penicillin succeeded in time to treat soldiers at the end of World manufacturing continues to evolve today variations in temperature, for example, War II (3). And biological with large-scale production of vaccines, will lead to cell death and eliminate an biopolymers, proteins, and viruses (4). entire culture. Loss of nutrients or Onlineanalysis is an essential part of accumulation of waste in a fed-batch PRODUCT FOCUS: BIOLOGICS modern manufacturing protocols. process will cause suboptimal reaction Currently, most biological manufacturers performance. Accumulation of excess PROCESS FOCUS: PRODUCTION use multiple analytical tools to gauge ammonia, for example, has been found WHO SHOULD READ: QA/QC AND critical process information suchas to interfere with protein glycosylation in PROCESS DEVELOPMENT osmolality, temperature, and pH. CHO cells (5). Biological systems are extremely sensitive The use and acceptance of nearKEYWORDS: PAT, SPECTROSCOPY, CELL complex matrices and require constant infrared (NIR) spectroscopy as an online CULTURE, BATCH PROCESSING data feedback, analysis, and control to multicomponent analytical tool forthe achieve a successful reaction. Small bioprocessing industry has grown LEVEL: INTERMEDIATE
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58 BioProcess International M ARCH 2008

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substantially since early studies of NIR in cell culture media appeared in the 1990s (6). One main reason for the popularity of Fourier-transform NIR (FT-NIR) as a measurement technique in cell culture is its ability to provide rapid,accurate, high-resolution chemical analysis for major components of interest such as glucose or ammonia, even when they are present at low concentrations. FT-NIR has been commonly used in industries such as chemical, polymer, and pharmaceutical manufacturing for decades, but its adoption has been slower for biopharmaceutical processes. Near-infrared spectroscopy uses overtones and combination bandsto show the concentration of certain analytes. Once light from an FT-NIR analyzer impinges on a sample, characteristic vibrational frequencies are absorbed by various molecular species (e.g., glucose or water), providing a unique spectrum. By collecting multiple spectra over time and combining them with known analyte concentrations, users can create a calibration using multivariate algorithms suchas partial least squares (PLS) regression. Such analytical technology allows for accurate, real-time predictions of multiple components within a complex matrix based on a single spectrum. Here we offer experimental data from a cell culture of HEK293 (human embryonic kidney) cells using a Thermo Scientific brand Antaris FT-NIR analyzer to predict four critical inprocess components: glucose,...