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Analytical Method Development & Validation

Analytical >> Analytical Method Development >> Biologic Drugs

AMRI specializes in biochemical method development, transfer and validation for the analyses of biological products and biosimilars by HPLC, UPLC, LC-MS, MALDI-TOF MS, NMR and assays.

Due to their complexity, biopharmaceuticals require a vast array of testing using orthogonal techniques. A tremendous amount of analytical testing is required to support a biopharmaceutical product from discovery, development, and clinical trials, through manufacturing and marketing. Numerous methods are used to fully characterize large molecules because of their complexity—characterizing them is significantly more difficult than it is for small molecules. Biopharmaceuticals are produced via living systems, i.e., E. coli, yeast, or mammalian cells, which require additional testing matrices. Further, FDA requires orthogonal techniques when available to better reveal the structure and stability of a biopharmaceutical. Currently, more than half of the drug candidates in the discovery stage are biologics, including proteins, peptides, monoclonal antibodies, etc. As the overall number of these large molecules in the pipeline increases, many drug manufacturers may need to consider outsourcing portions of their analytical testing to fill voids in capacity or to meet timelines. This article describes some of the analytical methods used to support a typical biopharmaceutical product throughout its lifecycle from discovery and early development through stability and release testing. Analytical Services for Biologics Stability Testing is used to demonstrate short and long term stability of a drug substance or product after exposure to a variety of environmental factors. Requirements of the ICH guidelines are met through our expertise to apply stability testing method to detect compound changes in identity, purity and potency. Stability Testing Services include: Alarmed monitoring systems, Secured storage areas, Temperature, humidity, and light variability, Labelling, handling and storage, Computerised entry and reporting, Long term stability testing, Accelerated stability testing. Reference Standard Characterization studies are used to determine the physiochemical profile of a drug substance or product in order to establish a standard for subsequent stages of testing. This testing is essential for characterising and analysing both original biologics and biosimilars. These studies often demand a broad variety of techniques and instrumentation for a complete analysis of drug purity, identity, and stability. Analytical testing program is customised to meet regulatory standards in the ICH guidelines Mass Spectrometry methods: Molecular Weight, Peptide mapping, Glycan Profiling, N-Terminal Sequencing UHPLC methods: Amino Acid Analysis, Glycan Profiling, SEC, IEX, RP chromatography, Extinction Coefficient Capillary Electrophoresis methods (CE, cIEF), Charge Profile, Isoelectric Point. UV Spectrometry, pH, Osmolality, Karl Fischer, SDS-PAGE, Western Blot analysis Lot Release and Comparability testing is necessary for evaluating the purity, potency, and identity of biologics. Analytical assays are available for quality control testing that is regulatory compliant. Product release testing is a crucial part of ensuring drug product quality and safety. Regulatory bodies such as the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) require lot release tests for each biopharmaceutical batch. This testing is performed at all stages of drug development:Raw Materials, Unprocessed Bulk, Purified Bulk (Drug Substance), Final Product (Drug Product) Lot Release Testing relies on previously established specifications to demonstrate product identity, purity, and potency. Similarly, Comparability testing is aimed at identifying whether changes in the manufacturing process lead to downstream changes in product quality. Method Validation is essential for adherence to Current Good Manufacturing Practice (cGMP) and Good Laboratory Practice (GLP). Analytical Method Validation (AMV) is a requirement within the biopharmaceutical industry for all methods used in testing raw materials, in-process materials, final containers and excipients. Method Validation is used to confirm that the analytical procedure employed for a specific test is suitable for its intended use and is required for complete compliance with cGMP and GLP regulations, US FDA and international regulatory guidelines. Some test method validation characteristics include: Optimization and development are largely completed prior to method validation studies Prior to validation testing, method performance is defined Requirements must be met by every validation trial run Method Validation needs to be performed in situations including: Method transfer to different laboratory Validation prior to initial use of method in ongoing or routine testing Validation upon any changes to previously-validated conditions or method parameters, if changes extend beyond scope intended by original method Phase Appropriate Validation characteristics include: Predetermined performance expectations of method Determine method performance elements such as specificity, linearity, accuracy and precision - for an intended application Various techniques commonly applied to biopharmaceuticals testing are Structural Elucidation and Characterization X-ray crystallography Nuclear magnetic resonance (NMR) Spectroscopy Ultraviolet spectroscopy (UV) Fluorescence spectroscopy Mass spectrometry (MS) Circular dichroism spectroscopy (CD) Fourier transform infrared spectroscopy (FTIR) Chromatography-based Techniques Reversed-phase, high-performance liquid chromatography (RP-HPLC) Ultra performance liquid chromatography (UPLC) Size exclusion chromatography (SEC-HPLC) Ion-exchange HPLC (CEX and AEX-HPLC) Gel permeation chromatography (GPC) Amino acid analysis (AAA-HPLC) Peptide mapping (HPLC) HPLC/MS Ion chromatography (IC) Gas chromatography (GC) GC/MS Aggregate Analysis Size exclusion chromatography-multi-angle light scattering (SEC-MALS) Field fIow fractionation-muIti-angle light scattering (FFF-MALS) Analytical ultracentrifugation (AUC) Elemental Analysis Graphite furnace atomic absorption (GFAA) Inductively coupled plasma (ICP) ICP-MS X-ray fluorescence spectrometry (XRF) Electrophoretic Techniques Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS—PAGE) Isoelectric focusing (IEF) Capillary zone electrophoresis (CZE) Safety, Microbiology, and Virology Testing Bacterial endotoxin involving inhibition-enhancement testing Bioburden Sterility including fungistasis-bacteriastatic testing Residual host cell DNA by qPCR Host cell proteins Mycoplasma Viral clearance Bovine spongiform Encephalopathy-transmissible spongiform Encephalopathy (BSE—TSE) In vitro adventitious virus assays Other tests Immunoassays Cell-based potency assays G-Protein coupled receptors (GPCR) assays