PROCESS ANALYSIS TECHNIQUES USED IN THE BIOTECH INDUSTRY
The demand for real-time and near-real-time monitoring in the past two decades has resulted in significant innovation and
automation in the field of process analyzers. In the following subsections, we briefly review some of the commonly used process
analysis techniques in the biotech industry (5).
Figure 1: Comparison of analyzers with respect to their ease of implementation in microbial fermentations and mammalian cell
culture unit operations for various quality attributes: (a) misincorporation, (b) nutrients, (c) glycosylation, and (d) cell
Near-infrared (NIR) spectroscopy is one of the most commonly used analyzers for PAT applications. It is based on molecular
overtone and combination vibrations. This analyzer typically utilizes a frequency range of 4000–12,500 cm-1 (800–2500 nm)
to cover overtones and combinations of the lower energy fundamental molecular vibrations that include at least one X–H bond
vibration. The functional groups involved in NIR (almost exclusively) are those involving the hydrogen atom: C-H, N-H, and
O-H. A key advantage that NIR has is the possibility of direct measurement of the sample (6,7) either in situ, or after extraction
of the sample from the process in a fast loop or bypass. The data from NIR measurements require multivariate analysis to extract
the desired chemical information (8). NIR spectroscopy and multivariate data analysis (MVDA) has been successfully used for
screening basal medium powders used in a mammalian cell culture in the biopharmaceutical industry (9) and also for at-line
control and fault analysis of high cell-density fermentations (10). NIR probes also are used in crystallization processes
to detect the particle size, shape, and the polymorphic form. This enables monitoring during routine production and determination
of the crystallization endpoint (11).
Raman spectroscopy is a spectroscopic technique used to study vibrational, rotational, and other low-frequency modes in a
system (12). A monochromatic light, usually from a laser in the visible, near-infrared, or near-ultraviolet range, interacts
with molecular vibrations and phonons, resulting in the energy of the laser photons being shifted up or down. The shift in
energy gives information about the compound in the system. Samples for analysis can be solids, liquids, gases, or any form
in between, such as slurries, gels, and gas inclusions in solids. The Raman spectrum of water is extremely weak so direct
measurements of aqueous systems are easy to do, giving this technique an advantage compared to infrared spectroscopy in which
water has a very strong absorption. There is no inherent sample size restriction because it is fixed by the optic probe (13).
Measurements can be made noninvasively or in direct contact with the targeted material (14). Applications in biotechnology
processing include the monitoring of moisture content during lyophilization (15).
Figure 2: Comparison of analyzers with respect to their ease of implementation in isolation and purification of biotech products:
(a) removal of cell mass, (b) misfolds, (c) charge variants; and (d) mass variants.
Nuclear magnetic resonance (NMR) spectroscopy exploits the magnetic properties of atomic nuclei to determine physical and
chemical properties of atoms or the molecules in which they are contained. It can provide detailed information about the structure,
reaction state, dynamics, and chemical environment of molecules which can be an essential tool for PAT (16). Duarte and colleagues
identified and characterized 30 compounds in beer through high resolution (HR)-NMR (17). Two-dimensional (2D) NMR spectroscopy
has been used for metabolic flux analysis of high-density perfusion cultures of Chinese hamster ovary (CHO) cells lines (18).
Bench-top (BT)-NMR has been used in characterization of emulsions and lipid ingredients and monitoring adsorption as a noninvasive
tool in drug delivery research (19).
Mass spectrometry (MS) uses the difference in mass-to-charge ratio (m/z) of ionized atoms or molecules to separate them from
each other. It is useful for quantitation of atoms or molecules and also for determining chemical and structural information
about them. Because of its inherent sensitivity, speed, and molecular selectivity, MS has been used for process analysis,
including in-process monitoring of exhaust gases of fermentation processes, monitoring of drying processes, and environmental
monitoring (20). Other applications include on-line, real-time deuterium abundance measurements in water vapor in aqueous
liquids, including urine and serum (21); real-time quantification of trace gases in food products (22); and obtaining structural
information for identification or structural elucidation of pharmaceutical drug products (23).