Acoustic resonance spectroscopy (ARS) involves spectroscopic measurements in the acoustic region, primarily the sonic and
ultrasonic regions. It is typically much more rapid than high performance liquid chromatography (HPLC) and NIR, and it is
nondestructive, requiring no sample preparation because the sampling waveguide can simply be pushed into a sample powder,
liquid, or solid (24). Applications include a high-throughput, nondestructive method of online analysis and label comparison
before shipping to obviate the need for recall or disposal of a batch of mislabeled drugs (25).
Figure 3: Comparison of analyzers with respect to their ease of implementation in formulation and packaging: (a) moisture
content, (b) product content, (c) foreign particles, and (d) labeling.
Calorimetry involves direct measurement of any endothermic or exothermic change in any process in order to better monitor
or control all chemical, physical, and biological processes by providing the ability to measure enthalpy, power, and the heat
coefficient (26). It is an on-line, nonintrusive technique for monitoring and optimizing a process. Applications include control
of cooling profiles or rates during crystallization, real time measurement of heat transfer coefficient, monitoring and control
of bioreactors, and determination of precise specific thermal profiles or signatures (27).
Dielectric spectroscopy is also known as impedance spectroscopy and measures the dielectric properties of a material. It offers
the ability to measure bulk physical properties of materials based on the interaction of an external field with the electric
dipole moment of the sample. It has an advantage compared to other spectroscopic techniques because it is not an optical spectroscopy
or a noncontact technique. This allows for measurement without disturbing a sample in process. This tool has been used as
a real-time and in-line monitoring tool for process optimization, automation, consistent product quality, and cost reduction
in mammalian and insect cell cultures (28).
Fluorescence spectroscopy is a type of electromagnetic spectroscopy that analyzes fluorescence by using a beam of light, usually
ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light at a lower energy
(29). Fluorophores such as proteins, coenzymes, and vitamins can simultaneously be detected qualitatively and quantitatively
inside and outside the cells. Cell metabolism and cell growth also can be measured (30). Teixeira and colleagues monitored
on-line cell viability of recombinant baby hamster kidney (BHK) cell lines, as well as the concentration of the expressed
glycoprotein IgG1-IL2 with 2D fluorometry and multivariate chemometric models (31). Other applications include real time monitoring
of cell density and antibody titer in bioreactors containing CHO cell lines for production of monoclonal antibodies (32) and
detection of antigen-antibody complexes for measuring immunoglobulin G (IgG) concentration in mammalian cell cultures (33).
On-Line HPLC: Rathore and colleagues have published a series of case studies that examine the use of on-line HPLC, at pilot
and manufacturing scales, for performing analyses to facilitate real-time decisions for column pooling based on product quality
attributes (34, 35). HPLC also has been used for the monitoring of protein purity and levels of the different protein-related
variants and impurities to enable the timely ending of refold on the basis of product quality data (36). The metabolism in
a mammalian cell culture was examined by monitoring the concentration of 17 amino acids and glucose with online HPLC (37).
Other applications include separation of recombinant human insulin-like growth factor-I (IGF) from IGF aggregate to allow
for real-time control of column pooling (38) and using a combination of on-line size-exclusion HPLC, differential refractometry,
and multiangle laser light scattering analysis (MALLS) for real-time estimation of product quality of a mutant form of the
human immunodeficiency virus (HIV) vaccine protein antigen (39).