1. Fermentation Overview
The pCI-neo Mammalian Expression Vector carries the human cytomegalovirus (CMV) immediate-early enhancer/promoter region to promote constitutive expression of cloned DNA inserts in mammalian cells. This vector also contains the neomycin phosphotransferase gene, a selectable marker for mammalian cells. The pCI-neo Vector can be used for transient or stable expression by selecting transfected cells with the antibiotic G-418.
Photo credit: courtesy Ahna Skop
CHO cells dividing. The cells are stained with anti-actin (red), anti-tubulin (green) and DAPI (blue). This image shows two Chinese hamster ovary cells in the last stages of division. The red outer membrane is complete around each new cell, while the green midbody still remains between them. Photo date: May 2004

2. Process Development
Take a few minutes to think about what steps would be necessary to develop a protein based medicine product?

3. Fermentation Pathway
A typical cell culture process comprises vial thaw, seed expansion, and production stages.

4. Generic Mab Process Cell Culture Centrifuge & Depth Filtration
Protein A Capture Column
Viral Inactivation
Cell Culture
Cation Exchange
Centrifuge & Depth Filtration
Drug Substance
Anion Exchange
Viral filtration
Ultrafiltration/ Diafiltration
Fill and Finish

5. General steps in Process Development for a Protein Based Medicine
Drug target selection
Expression system and host ( R&D)
Basic media and culture optimization, choose lead clone(s)
Fermentation process (Upstream Process)
Clarification system (Midstream process)

6. General steps in Process development
Downstream isolation and purification
Formulation of final drug product and stability testing
Bioanalytical testing, Meet target product profile (TPP)
QA/QC, and regulatory affairs
Other points, i.e. cell banking, process robustness, viral clearance, toxicity studies, technology transfer

7. Proteins as Biotechnology Products
Making a Biotech Drug
Produced through microbial fermentation or mammalian cell culture
Complicated and time-consuming process
Must strictly comply with regulatory agencies at all stages of the process.

8. Recombinant proteins

9. Expression system and Host
Related, need to know host to figure out best expression system, bacteria, fungi, plant, mammalian, etc.
E. coli, bacteria
A. niger, fungi
mammalian ie CHO, NSO

10. Host selection
Simple molecules can be expressed in bacteria plants or fungus more efficiently and cheaper
Complex molecules like antibodies with glycosolation may need a mammalian host
Chinese Hamster Ovary (CHO) cells are the most frequently used, grow fast and produce well.
Plus have already had regulatory approval!
Human cells sometimes used, such as HEK 293 cells (embryonic kidney)

11. Protein Modifications
Glycosylation – post-translational modification wherein carbohydrate units are added to specific locations on proteins.
More than 100 post-translational modifications can occur.

12. Glycosylation reactions in the Golgi
Thorsten Marquardt Nature Medicine  10, (2004)

13. Different organisms have different Glycosylation patterns

14. Mammalian DMEM media COMPONENTS – Many ( >80) and Well defined!
Inorganic Salts CaCl2, Fe(NO3)3 • 9H2O, MgSO4, KCl, NaHCO3, NaCl, NaH2PO4
Amino Acids L-Alanyl, L-Glutamine, L-Arginine • HCl, L-Cysteine • 2HCl, L-Glutamine, Glycine L-Histidine • HCl • H2O, L-Isoleucine L-Leucine, L-Lysine • HCl, L-Methionine, L-Phenylalanine, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine • 2Na • 2H2O, L-Valine
Vitamins Choline Chloride, Folic Acid, myo-Inositol Niacinamide, D-Pantothenic Acid • ½Ca, Pyridoxal • HCl Pyridoxine • HCl, Riboflavin, Thiamine • HCl
Other D-Glucose, HEPES, Phenol Red • Na, Pyruvic Acid • Na, Trace elements
Mammalian media is a complex media with >90 components, and are well defined. Bacterial and Yeast media have only a hand full of components that are not well defined

15. General Fermentation Batch – No feeding of the culture
Fed Batch – Feeding of the culture
Perfusion – Simultaneous feeding and removal of the culture
Process for Antibody production reach 1-10 g/L
Best process typically not the most robust, manufacturers will take 10-20% reduction in productivity to achieve a reproducible process

16. Necessary work before production
Select molecule to produce (DNA sequence) R&D
Expression vector (Plasmid) R&D
Host for protein expression, bacteria, yeast, mammalian cells, etc. R&D
Pick lead clone to work with R&D and Fermentation
Optimize growth and feed media Fermentation
defined media is best, no serum if possible
Optimize seed train, Fermentation
typically 5x-10x culture expansion each step

17. Consistency of Process is KEY!
Need to get approval from regulatory bodies, FDA, EMA, etc.
The end process must meet cGMP standards!
Need consistent process for consistent product
Need stability testing of system.
Start from Master Cell Bank (MCB)
Progress through seed train
Then off to large scale fermentation

18. Fermentation Time Table
Ferm vessel
Volume (L)
Time (days)
Total time –days
MCB thaw
0.05
Shake flask 1
0.1 3
Shake flasks 2
0.4 6
Shake flasks 3
1.5 9
Wave bag 1 5 12
Wave bag 2 20 15
Reactor 1
100 18
Reactor 2
500 21
Reactor 3
2500 24
Production reactor
12500
~13
~37
Typical CHO fermentation for Antibody process
Assumes cells with a 24 hour doubling time and ~ 4-5 x expansion per step

19. Master Cell Bank (MCB) Why use MCB? Typically 250-500 vials
Store in Multiple locations
Need consistency in process!
Stability of cell growth, productivity and product profile
Heavily tested!!
Viruses, growth, product quality, etc.
Some groups then create Working Cell Bank (WCB)
Once again typically ~ vials

20. Growth Curves Plot the total viable cells at any time in the culture
Typically expressed as 10e6 viable cells/mL
IVCD (integral of viable cell density)
Area under the curve
Want to maximize IVCD of culture, Why?
Viable cell curve
Calculating area under the curve

21. Specific Productivity
Each clone produces a characteristic amount of product (ie antibody)
Expressed as Picogram (protein)/cell/day (PCD)
When choosing a lead clone, want to balance good specific productivity (PCD) with good growth (Max IVCD)
PCD X IVCD= mg/L of protein produced (for antibodies)
Example: 40 PCD and 50 IVCD = final culture with 2000 mg/L antibody concentration

22. Bioreactor Basics

23. Seeding Production Reactor
To few cells takes to long with lag phase growth
To many cells have carry over of metabolites and leads to lag phase too.
For CHO system optimal inoculation is ~10-20 % of final culture volume.
Typically seed between 0.15 and 0.4 x 10e6 Viable cells /mL
For a bacterial system only need ~1% of final culture volume for seeding

24. Bioreactor Parameters
Dissolved Oxygen – needed for cell growth and metabolism
To little and cells will be oxygen starved, reducing growth and impacts on product characteristics
To much leads to oxygen stress of the cells and oxygen radicals and increased DNA damage
Typical setting range from 20-70% dissolved oxygen
Measure this on line with DO probes in bioreactor

25. Bioreactor Parameters
Agitation – Need to keep the cells in suspension and well mixed for access to food and oxygen
To little agitation and cells can settle and can not adequately access food and may start to clump or have growth inhibition.
To much agitation leads to mechanical shearing and damages cells. Adding certain chemicals can help minimise shear effect ie. Pluronic F-68 at 0.1%.
Agitation rate needed depends on size of impeller used
Slower RPM with larger diameter
Pluronic F-68 at 0.1% can assist in minimizing shear effect

26. Bioreactor Parameters
Temperature – Need to optimize temperature for the system
For E. coli and most mammalian systems temperature optimum is ~37 °C
Can temperature shift lower at times (32-35 °C) once cell numbers nearly reach the plateau to change from cell growth to protein production.
Temperature shift can lengthen culture life (increase IVCD) by arresting cells in G1 phase of cell cycle.

27. Bioreactor Parameters
pH – optimizing pH can have critical impacts on productivity
Typically in pH 6.8 – 7.2 range
Measure on line with probe in bioreactor
Lowering pH can slow down cell growth and productivity
Increasing pH can raise productivity and increase cellular metabolism as well as waste products
Can alter pH at various times in the culture to optimize protein production

28. Fed batch fermentation
Fed Batch - feeding additional components to the bioreactor during the course of the fermentation.
This typically improves cell numbers and product yield, but adds complication to process.
Main additives for mammalian system
Glucose
Glutamine
Starting media concentrations
Glucose – 6-8 g/L
Glutamine 6-10 mM

29. Fed batch fermentation
Glucose or glutamine starvation.
If starved, cells stop growing, reducing product production
Reaching 0 g/L glucose or 0 mM glutamine is very bad!
Do not want to over feed or cells will metabolize extra glucose and glutamine, leading to a build up of waste products, ie. lactate and ammonia which inhibits cell growth and product production.
Over 8-10 g/L glucose and 8-10 mM glutamine leads to problems with cell growth and viability

30. Media Analysis Need to check various paramaters of your media
pH, Glucose, Glutamine, Ammonia, Lactate, Dissolved O2, CO2, Potassium, Phosphate, Antibody conc. etc,
Typically done with automated analysers
NOVA flex, BGA, etc.

31. Typical pH and lactate profiles in a fed-batch process

32. Assessing Cell numbers
Manual counting methods, Hemacytometer
Automated methods – Vi-Cell, Countess, CEDEX, etc
Need to know cell numbers to understand ferment
Hemacytometer
Vi-Cell

33. Assessing cell viability
Want to keep cell viability as high as possible for as long as possible
Trypan blue assay – dye exclusion method (Vital dye)
Live cells with intact membranes exclude dye
Dead cells take up dye and are stained dark blue
Know health of culture
Trypan blue

34. Genentech video
Quick overview of Genentech with Herceptin ab product

35. Stainless Steel vs Disposable
Stainless advantages
Can use over a long time, years
Can make at any scale
Stainless disadvantages
Cleaning costs
Slow turn around time
Minimal flexibility once installed
Disposable advantages
No cleaning and validation costs
Rapid turn around time
Flexible footprint
Disposable disadvantages
Greater cost in long run
Limited size (up to ~ 2000 L) at current time

36. Conclusions
Recombinant proteins can be made in a number of different hosts.
Some proteins need mammalian cells for proper function (glycosylation)
Drug target selection can be difficult
Lots of risk
Expression systems vary and you need to know host and product for informed choice

37. Conclusions II
Need to select a good lead clone for manufacturing, so you need good ones to choose from!!!
Sounds simple, but not always the case!
Need to optimize pH, Temperature, Oxygen levels, agitation for optimal process
Need to optimize media and feed parameters in a fed batch process.
Need to constantly monitor cell culture and media for best results.

38. Pharmatopia lab
Next you will apply your gained knowledge in a fermentation practical simulation in Pharmatopia
Biotechbasic:  
Biotech advanced: