1. Northeast Biomanufacturing Center and Collaborative
Process Development
Northeast Biomanufacturing Center and Collaborative

2. 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

3. 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

4. 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

5. 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

6. Stages of Process Development
Early stage
Mid-stage
Late stage

7. 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

8. 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

9. 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

10. 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)

11. Example – In Process Hold Study

12. 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

13. 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

14. 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

15. 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