CENG 109 class 5

Bioprocess Engineering

Class announcement
Class project
Team forming situation Schedule for library training will be posted on class web site. Attendance is mandatory

Make-up class in early October (to replacing missing class on October 24)

Outline for this class

The definition and scope of bioprocess engineering The technical challenges for bioprocess engineers Case studies of early biotechnology products
-Insulin -tPA

The economic impacts of bioprocess engineering

Science and the application of science are linked as the fruit is to the tree
Louis Pasteur

Definition of Bioprocess Engineering

Discovery in Basic Science
Market Force Bioprocess Engineering Practical Products, Processes or Systems

What is bioprocess engineering about? A bioprocess includes an upstream process and a downstream process
Upstream: product production in cell cultures/fermentations Downstream: isolation of product and its formulation

What is bioprocess engineering?

the design and construction of a bioreactors (fermentation tanks) to grow cells the design and utilization of different equipments to isolate the products traditional engineering approaches for scaling up and minimizing costs the design and construction of living organisms to optimize product production

Technical Challenges
Number of protein products: from almost none to many Complexity of protein products: High Liability of protein products: High Purity requirement: High Scale of production and product pricing: Highly variable

Unit value and production quantities of early biotech products

Product Approx. selling price Amount of Product $/g for $200M in sales, kg 20,000 530 5.7 8.7 0.24

Human serum albumin 10 Human insulin Growth Hormone Tissue plasminogen activator (tPA) Erythropoietin 375 35,000 23,000 840,000

Source: National Academy of Sciences, Committee on Bioprocess Engineering (1992)

Insulin case study

The first production process of human insulin involved separate expression of A and B chains. To stablize each chain, each chain is fused to a larger protein to form insoluble granules. The fusion proteins are subsequently removed and the two chains recombined .

Figure extracted from Bioseparations Engineering: Principles, Practice and Economics by Ladish

Upstream process made more efficient by mimicking nature

This figure Illustrates insulin synthesis in pancreas

The proinsulin method is adopted to improve production efficiency

Figure extracted from Bioseparations Engineering: Principles, Practice and Economics by Ladish

The proinsulin method is adopted to improve production efficiency

The one-chain proinsulin method has been adopted to improve the yield of correctly folded proteins. What kinds of expertise are required to achieve this improvement?

Isolation of human insulin requires 27 steps

Why multiple steps are needed? Principles of orthogonality Product is the minority, to be selected among many different biomolecules and cell debris Similarity bewteen product and Junks Regulations require high product purity (contaminants below parts per million range)

Figure extracted from Bioseparations Engineering: Principles, Practice and Economics by Ladish

Product yield loss amplified by the number of purification steps

Overall Product Yield = (1- L%)n x 100% Lets calculate the yield for a 10-step process with a step loss of 5% The early insulin production process has a overall yield of 29% => Lower product yield is translated to higher manufacturing cost

tPA case study

Tissue plasminogen activator can dissolve blood clots --- is a therapeutic for treating heart attacks and strokes Sold as Activase, its sale accounted for 30% of total sales of GENENTECH, and about 1/3 of the worldwide sales in blood clot dissolving therapeutics in 1995 Unlike insulin, it is regulated as a biologic, not a drug

Production of tPA: a unique opportunity for the application of recombinant technology

50,000L of blood required to extract single dose of tPA (~100mg) not practical Recombinant technology provides a feasible approach

But tPA is much more complicated than insulin

Insulin Size Disulphide bonds Glycosylation sites tPA 5,600 Da 66,000 Da 3 None 12 4

.and achieving correct protein folding is more difficult

Molecular arrangement of tPA

Lets count the no. of disulphide bridges (solid bars), potential cleavage sites (arrows), glycosylation sites (zigzag lines) and short peptide bridge (dotted line)
Figure extracted from Bioseparations Engineering: Principles, Practice and Economics by Ladish

Upstream process can be made more efficient by mimicking nature

Bacteria (E.Coli) Replication rate Maintenance cost Remove introns? Posttranslational modification? Secrete products to medium? High Low No None No Mammalian Cells (e.g. Chinese Hamster Ovary Cells) Low High Yes Extensive Yes

Mammalian cell culture creates new downstream challenges

Fear of transfer of infectious agents Fear of genetic materials from cell lines causing cancer Purification process must be able to remove cells, virus and DNA Analytical assays must be able to detect very low level of cells, virus and DNA Lead to advance in downstream processing units, particularly membrane and chromatography applications

Get it fast and get it right the first time

Bioprocess cost is secondary to being first to market But cost of product is important in subsequent competition with rival products For biologic, the license provision requires not just the product, but also the manufacturing process/facility be approved A huge cost barrier exists to implement process changes after license is granted