1. Quality Control Biochemistry
Northeast Biomanufacturing Center and Collaborative

2. 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

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

4. QC Biochemistry Responsibilities
Maintain cGMP 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

5. 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 ( CoA) 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

6. Example of a Drug Product Certificate of Analysis

7. 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!!!

8. 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

9. Example of Specification Table of Hypothetical monoclonal antibody drug substance

10. 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

11. Analytical Method Life Cycle

12. Evolution of QC Methods & the Lifecycle of a Biotechnology Product

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

14. Method Development & Qualification
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

15. 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

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

17. 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
Osmolality – the number of osmoles of solute particle per kg of pure solvent (mOsm/kg)

18. Compendial Methods pH – pH meter and NIST pH 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
Osmolality – the number of osmoles of solute particle per kg of pure solvent (mOsm/kg)

19. 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
Osmolality – the number of osmoles of solute particle per kg of pure solvent (mOsm/kg)

20. 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 280nm, colorimetric assays (Bradford, BCA)
Purity – confirm the purity of the API
Chromatography (HPLC, Affinity, Size Exclusion)
SDS-PAGE

21. 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

22. 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

23. TYPES OF ELISA Direct ELISA- Indirect ELISA Competitive 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
Indirect ELISA detects the presence of a certain antibody in a sample (serum)
antigen is coated on the plate, then sample is put on (antibody will bind if present), then add enzyme-conjugated antibody to detect

24. Types of ELISAs

25. 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