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).

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.

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).

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).

In situ microscopy (ISM) involves the use of a direct-light microscope with a measuring chamber, integrated in a 25-mm stainless steel tube, as well as two CCD-cameras and two frame-grabbers. The data obtained are processed by an automatic image analysis system. The microscopic examination of the liquid is performed in the measuring chamber, which is situated near the front end of the sensor head. ISM mainly allows for on-line microscopic observation of microorganisms during fermentations in bioreactors (40). Applications include in-line monitoring of Hansenula anomala culture in a batch fermentation process; real-time monitoring of hybridoma cells concentration in a stirred bioreactor; measurement of the level of colonization of fibroblasts (murine cell line NIH-3T3) on microcarrier surfaces during cultivation; detection of cell viability without conventional staining techniques; and on-line monitoring of enzyme carriers to determine their mechanical stability in the bioreactor (41-45).

Microwave resonance technology (MRT) is a noninvasive and nondestructive method of evaluating the moisture content of analytes using a microstrip waveguide with a resonance in the 300 MHz to 3 GHz range. It has been used for continuous moisture measurement in pharmaceutical granules and for determining moisture, temperature, and density of the granules to improve process understanding in fluid bed granulation (46).

Terahertz technology has a frequency gap from 100 GHz to 30 THz in the electromagnetic spectrum, which is in between microwave and infrared and has found a variety of applications in biology, medical science, quality control of food, pharmaceutical, and agriculture products, as well as global environment monitoring. Terahertz time domain spectroscopy (THz-TDS) also has been applied to many materials, including biomolecules, medicines, cancer tissue, DNA, proteins, and bacteria (47). It enables 3D imaging of structures and materials, and the measurement of the unique spectral fingerprints of chemical and physical forms, which is especially relevant in 3D imaging of tablets (48) and estimation of concentrations of the various excipients (49).

Photoacoustic spectroscopy (PAS) is based on the absorption of electromagnetic radiation by the molecules in the sample. The nonirradiative collisions of molecules in sample lead to warming of the sample matrix, which gives a pressure fluctuation because of thermal expansion that can be detected in the form of acoustic waves (50). These waves propagate through the volume of the gas to the detector (microphone, piezoelectric transducers, or optical method) where a signal is produced. This detector or microphone signal, when plotted as a function of wavelength, will give a spectrum proportional to the absorption spectrum of the sample (51). Applications include on-line and nondestructive monitoring of growth, thickness, and detachment of biofilms in waste water treatment plants and unraveling the influence of various process parameters (such as pH, flow conditions) on the structure and stability of a biofilm (52).