Quality Control Biochemistry Northeast Biomanufacturing Center and Collaborative

Roles of Quality Control Biochemistry § Primary responsibility is to provide scientific evidence to ensure that products are consistently produced to meet the purity, safety, and efficacy standards § Test, measure, and evaluate biopharmaceutical drug substance from a production run against a specification for that substance

QC Analyst Skills and Abilities § High level of integrity § attention to detail § trustworthiness and reliability § Good communication skills

QC Biochemistry Responsibilities Maintain c. GMP laboratory - general lab“ housekeeping” § all equipment is within its calibrated use period § all solutions, standards and reagents are labeled and stored appropriately and within expiry period § all equipment is performing adequately Maintain QC laboratory documentation system SOP’s, forms, training records periodically reviewed and updated

Additional Responsibilities Generate testing data and perform data trending § Routine release and stability testing § test process intermediates, drug substance, drug product and samples from product stability studies § run defined sets of tests and evaluate using predefined acceptance criteria/specifications and generate Certificate of Analyses ( Co. A) for product § Evaluate test data for trends to monitor how a process performs over time Conduct QC related investigation § identify and investigate unexpected test results or problems (deviations) § assess impact of issues/deviations using various analytical methods and supports appropriate corrective actions

Example of a Drug Product Certificate of Analysis

Specifications § Set of limits that allow analyst to determine if tests meets pre-defined acceptance criteria § Development of specifications is integrated with the product development • Based upon scientific principles and intended therapeutic use • Regulatory requirements for quality and stability – Product lot meets the specification for the associated tests- batch “passes” and can be released – When batch fails to meet specifications the batch can not be released and the failure must be thoroughly investigated – Failed batch can result in lost time and money!!!

Types of Specifications Raw material specifications • control quality of incoming raw materials In-process specifications • control quality of in process intermediates Release specifications • control quality of both the Active Pharmaceutical Ingredients (API) and the final drug product

Example of Specification Table of Hypothetical monoclonal antibody drug substance

Analytical Methods Laboratory procedures used to measure a physiochemical or biological entity, or product attribute to verify its structural quality, purity and biological activity and its composition in the formulation § Developed and implemented through an evolutionary process (life cycle) § begins in development and continues through validation and production § as product goes through clinical development, requirements for scientific rigor, robustness of method and compliance increase in importance

Analytical Method Life Cycle

Evolution of QC Methods & the Lifecycle of a Biotechnology Product

Life Cycle of a Method Three (3) main stages in the life cycle of an analytical method • Method Development • Method Qualification • Method Validation

Method Development & Qualification Method Development • process that generates a measurement scheme that consistently provides a reliable result Method Qualification • set of experiments which demonstrate that an analytical method performs as expected and provides consistent and meaningful data under a defined set of conditions

Method validation Creates documented evidence that provides a high degree of assurance that a specific analytical method will produce data that consistently meets analytical performance parameters as they apply to the intended use of the method Includes performance criteria for the following parameters • • • Accuracy and precision Specificity Linearity Range Limit of Detection Limit of Quantitation

Data Trending –Figure 9 -12 Illustration of data showing the trending of cell viability across different assay runs over time

Types of Analytical methods Methods which determine the identity, purity, potency (bioactivity), and an evaluation of impurities are required by regulatory agencies to approve the release of a drug product. These methods fall into two major categories § Compendial test methods § Standards published in pharmacopeia (United States Pharmacopeia, European Pharmocopeia, and Japanese Pharmacopeia) § Non-compendial test methods § Often developed in-house and are typically specific to a particular product

Compendial Methods • p. H – p. H meter and NIST p. H control standards • osmolality – # moles soluteosmometer that uses freezing point depression; NIST standards • appearance – visual inspection-color, clarity and visible particulate matter; turbidimeter to determine clarity • content uniformity – 10 random vials, reconstituted and concentration for each vial is calculated to determine diff b/w label claim and vial conc

Compendial Methods Continued • bacterial endotoxin – endotoxin analysis system • sterility – no growth of microbial bacteria or fungal organisms • residual moisture content – colorimetric detection of water • Sub divisble particulate matter- assessed for final drug product using a light obscuration particle count test

Non-Compendial Methods Identity – confirm the presence of the API • ELISA • Ion exchange chromatography • peptide mapping Content – assess that a drug product is at the correct concentration for clinical dosage • Protein concentration – absorbance at 280 nm, colorimetric assays (Bradford, BCA) Purity – confirm the purity of the API • Chromatography (HPLC, Affinity, Size Exclusion) • SDS-PAGE

Non-Compendial Methods Continued Potency – biological activity • cell-based assays measuring cell proliferation, inhibition of cytotoxicity Impurity/residual tests – host cell proteins, residual host cell DNA Characterization • Carbohydrate Profile • peptide mappings Stability indicating methods - differentiate between unaltered drug and possible degradation products

ELISA Enzyme Linked Immunosorbent Assay – Immunodetection- Use antigen (Ag) and antibody (Ab) interaction to determine the concentration and/or the activity of a protein of interest – Read on a microtiter plate reader – a mini-spectrophotometer that determines the absorption or transmission of a beam of light of a particular wave length passing through a solution of the protein of interest – Using standards to generate a standard curve the concentration of the protein of interest in a sample can be determined

TYPES OF ELISA • Direct ELISA • primary Ab directly labeled with detection reagent • not commonly used for immunoassays • used for immunohistochemical staining of cells and tissues • Indirect ELISA • more commonly used in analytical assays • primary Ab binds to Ag immobilized on microtiter plate. Ag is detected indirectly by using a labeled secondary Ab to primary Ab • e. g. - Sandwich assay- Ag is sandwiched between bound capture Ab and detection Abs; more sensitive because of signal amplification • Competitive ELISA – for antigen specificity – unlabeled Ags compete with labeled Ags for binding to bound Ab

Types of ELISAs

Electrophoresis Polyacrylamide gel electrophoresis (PAGE) • used to determine purity of products( mostly proteins) after separation/purification steps • Separates molecules on a polyacrylamide gel matrix when an electric field is applied SDS-PAGE • Sodium dodecyl sulfate (SDS), a negatively charged detergent binds and coats proteins with negative charges • Negatively charged coated polypeptide chains then separate by molecular mass and not charge when an electric field is applied