Common Analytical Techniques Used for Residual Impurity Analysis

Mass spectrometry (MS) is an analytical technique for the determination of the elemental composition of a sample or molecule. It is also used for elucidating the chemical structures of molecules, such as peptides and other chemical compounds. The MS principle consists of ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios. MS instruments consist of three modules: an ion source, which converts molecules into ions; a mass analyzer, which sorts the ions by their masses by applying electromagnetic fields; and a detector, which measures the ions thus, providing data for calculating the abundances of each ion present. In a typical MS procedure, the sample is introduced to the MS instrument after chromatographic separation (GC or LC). The components are ionized by one of a variety of methods (electron impact, electrospray, or chemical ionization). The ions are then directed by an electromagnetic field in an ion trap, quadrupole, or time-of-flight to a detector. Computation of the mass-to-charge ratio (m/z) is then performed to generate a mass spectrum.

MS has both qualitative and quantitative uses and is one of the primary tools for monitoring and identifying residual impurities. Extractables and leachables profiles are typically generated using a combination of GC/MS, LC/MS, and ICP/MS. Triple quad mass spectrometry is a powerful tool for monitoring known residual impurities. Tandem quadrupole mass spectrometers separate a compound, and further break it down to yield highly specific daughter ions. These daughter ions can be used for quantification and yield high sensitivity and selectivity. Residual antibiotics can be measured using LC/MS/MS. Internal standards are commonly used and in many cases, part-per-billion (ppb) levels can be accurately detected and quantitated in very complex sample matrices.

Gas chromatography (GC) is another one of the most common methods of separation and analysis. Separation of components of a mixture is achieved by taking advantage of their different solubility. Therefore, the partition of the solute between the gas phase and the liquid phase coating the chromatographic column provides the basis of the analysis. Samples are introduced into the GC column either by direct injection of the liquid or by sampling the headspace over a liquid sample. The most common detectors include flame-ionization and mass spectrometer. The technique is best suited for volatile and semivolatile organic compounds and is commonly used for residual solvent analysis.

Ion chromatography (IC) is a process that allows the separation of ions and polar molecules based on the charge properties of the molecules. IC can be used for almost any kind of charged molecule including large proteins, small nucleotides, amino acids, and small molecules. It is a powerful technique best suited for determining low concentrations of ions.

Inductively coupled plasma (ICP) is a technique where samples are vaporized in an argon plasma. These instruments can be coupled to an optical emission or mass spectral detector. They are used to determine the level of various metals.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) is a denaturing gel electrophoresis technique used in the development and characterization of proteins. SDS is a detergent that dissociates and unfolds oligomeric proteins into its subunits. The SDS binds to the polypeptides to form complexes with fairly constant charge to mass ratios. The electrophoretic migration rate through a gel is therefore determined only by the size of the complexes. Molecular weights are determined by simultaneously running marker proteins of known molecular weight. The gel can be stained by silver staining or colloidal blue. The gels are scanned and analyzed using a densitometer to identify the sample bands. This approach can be used to monitor HCP.

Western blot is an analytical technique used to detect specific proteins in a given sample. Blotting is the transfer of large molecules on to the surface of an immobilizing membrane. This blotting technique is used to establish protein identity and purity. Native or denatured proteins are separated by gel electrophoresis and then transferred to a membrane. They are then identified using antibodies specific to the target protein, which develops color at the site of the protein-antibody complex on the membrane. This approach also can be used to monitor HCP.

Polymerase chain reaction (PCR) is a technique used to amplify a single or few copies of a DNA fragment by several orders of magnitude when the ends of the sequence are known. Genomic DNA is digested into large fragments using a restriction enzyme and then is heat-denatured into single strands. Two synthetic oligonucleotides complementary to the 3' ends of the target DNA segment of interest are added in great excess to the denatured DNA, and the temperature is lowered to 50–60 °C. The genomic DNA remains denatured because the complementary strands are at too low a concentration to encounter each other during the period of incubation but the specific oligonucleotides, which are at a very high concentration, hybridize with their complementary sequences in the genomic DNA. The hybridized oligonucleotides then serve as primers for DNA chain synthesis, which begins after addition of a supply of deoxynucleotides and a temperature-resistant enzyme DNA polymerase (Taq polymerase). Taq polymerase can extend the primers at temperatures up to 72 °C. When synthesis is complete, the whole mixture is heated up further (to 95 °C) to melt the newly formed DNA duplexes. When the temperature is lowered again, another round of synthesis takes place because excess primer is still present. Repeated cycles of synthesis (cooling) and melting (heating) quickly amplify the sequence of interest. At each round, the number of copies of the sequences between the primer sites is doubled, and therefore, the desired sequence increases exponentially. PCR is a great technique for confirming residual DNA clearance. Another form of PCR called reverse-transcriptase (RT-PCR) can be used for residual RNA.

Enzyme-linked immunosorbent assay (ELISA) is used to detect an antibody or antigen in a sample. The sample with an unknown amount of antigen is immobilized on a solid support either by adsorption to the surface or by capture methodology, known as "sandwich" ELISA. After the antigen is immobilized on the support surface, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody, which is linked to an enzyme through bioconjugation. After washing, the plate is developed by adding an enzymatic substrate to produce a visible signal, indicating the quantity of antigen in the sample. There are kits available for HCP specific to a given cell line.