Northeast Biomanufacturing Center and Collaborative Process Development Northeast Biomanufacturing Center and Collaborative

Process Development Objectives To develop a robust, scalable, reproducible, and cost-effective process that results in safe and efficacious biopharmaceuticals Time consuming and expensive 8-15 years $500 million to $1 billion Begins when a potential drug is first expressed in a cell and is proven to have the correct identity Evaluation and optimization of various steps in identifying and purifying the product Scale up to manufacturing

Choice of Expression System Two main criteria: protein structure – whether or not it has a post-translational modification cost of goods – the costs of different systems can vary greatly Post-translational modification Modification of an amino acid of the polypeptide chain Can be additions of other side components Lipid or oligosaccharide (lipoprotein or glycoprotein) Oxidation, de-amination, cross-linking, etc. Most important is glycosylation Addition of sugar moieties to certain amino acid side chains Approximately 70% of human proteins are glycosylated Biological activity is highly affected

Types of Expression Systems Bacterial Systems inexpensive unable to perform post-translational glycosylation Yeast Cultures add non-human glycans which are immunogenic Insect Systems occupy space between yeast and mammalian systems both in cost and characteristics not common used

Types of Expression Systems Contd Mammalian Systems widely used expensive and complicated Chinese Hamster Ovary cells (CHO) and non-immunoglobulin secreting mouse myeloma cells (NSO) Transgenic Systems expression in both animals and plants favorable cost flexibility and simplicity of scale up

Stages of Process Development Early stage Mid-stage Late stage

Process Development Early Stage Mainly research Goal is to express a recombinant protein with a well-defined biological activity Objective is to prove that the expressed protein contains the correct sequence of amino acids No regulatory restrictions apply at this stage

Process Development – Mid-Stage Most of the development activities occur during this stage Process characterization and process validation studies Preclinical and clinical evaluation Analytical tools developed to assess the result of the development activities Tools address biological activity, product identity, product purity and product and process-related impurities Product impurities – truncated and aggregated product, incorrect disulfide formation, and other chemical changes of certain amino acid residues Process-related impurities - host cell proteins, growth medium or buffer components that might be hazardous if in final product SOP’s and Batch Records written and approved

Process Development –Late Stage Begins with the filing of the Biologics License Agreement (BLA) Preparations are made to begin Clinical Phase III study Process validation begins after process development is complete

Process Characterization Involves performing bench-scale studies to demonstrate process robustness and help predict the performance of the process within the constraints of the operational ranges to be used Operating parameters (OPs) are identified that are most likely to impact the Process Performance Parameters (PPs) Biochemical study – focus will be on the integrity of the product Aggregation - Size Exclusion Chromatography (SEC) Degradation -SDS-PAGE under reducing and non-reducing conditions Isoform analysis - Isoelectric Focusing (IEF) Solution holding studies (growth medium and buffer)

Example – In Process Hold Study

Process Validation Provides documented evidence that a given process, operated within established parameters, can effectively and reproducibly produce an Active Pharmaceutical Ingredient (API) meeting predetermined acceptance criteria Establish appropriate Validation Acceptance Criteria (VAC) One of the greatest challenges in the development of a biopharmaceutical manufacturing process Traditional process validation consists of nine runs for the validation of a parameter – parameter tested at low, mid and high point for each parameter, with each point tested three times

Process Validation Studies Small scale studies: Growth media and buffer stability (chemical and/or biochemical) Collect data on how long the components of a solution are stable Initial resin and membrane reuse and hold studies Assess how long the chromatography resins and cross-flow membranes can be stored how many cycles of production matrices can take without loss of performance Process and product-related impurities Clearance studies – host cell DNA, endotoxins, viral clearance Removal studies – certain media components, leaching protein ligands from resins, leachables and extractables from single-use filters and media bags, hazardous chemicals used in the process Product-related impurities Truncated, aggregated, modified Full Scale Studies Media and buffer storage expiration studies Assessing bioburden in the actual storage vessels Resin and membrane reuse and cleaning/storage studies

Viral Inactivation and Clearance Potential risk associated with the production and use of biological products is viral contamination Present in source material or introduced in the manufacturing process through the use of products of animal origin in cell culture Studies must cover all known and potentially unknown types of viruses

Viral Inactivation and Clearance Studies Inactivation Studies Performed to find conditions that will inhibit virus reproduction while leaving product unharmed Methods include pH adjustment or solvent/detergent treatment Clearance Validation Studies To establish, ideally both qualitatively as well as quantitatively, the overall level of virus clearance Evidence must be obtained in all stages of purification, and adequate viral removal and/or inactivation must be proven Method is to challenge the system through the deliberate addition (spiking) of significant amounts of virus