1. Chapter 10 - Upstream Processing
Northeast Biomanufacturing Center and Collaborative

2. Upstream Processing Overview
Main objective – to create the environment necessary for cells to make a protein product (recombinant protein or biologic)
Product of interest
Called API’s- Active Pharmaceutical Ingredient
Produced by mammalian cell culture (Chinese Hamster Ovary - CHO cells, mouse myeloma NSO cells) or bacterial fermentation (E.Coli)
Mammalian cells excrete product into media
can modify proteins (post translation modifications)
harvest can be made without cell lysis
CHO cells most commonly used
Bacterial cells
do not have the machinery to secrete the desired product into the media
cells must be lysed during harvest

3. Biomanufacturing Process Flow – Upstream Processing

4. Upstream Processing - Cells
Cells used in upstream processing:
contain the transfected gene that expresses the desired API (protein)
transfected gene is linked to a gene that imparts special survival abilities to the cells that have it
kept in cryovials in Dewars
Dewar – a specialized vessel that provides the environment to keep cell processes in temporary frozen suspension
Cells in upstream process have been through intense study for many years

5. Upstream Processing Areas, Equipment & Systems
Dispensing room
CIP/SIP systems
Media Preparation area
Cell Culture/Fermentation
Cell Banking Area
Bioreactors
Primary Recovery (harvest)

6. Dispensing Room
Materials needed to make product are weighed and measured
Environment and access are strictly monitored traceably documented
Containment and Access
Air quality and flow
Cleanliness

7. Containment and Access
Containment booths to prevent cross contamination of one material to another
non animal derived and animal derived materials segregated to reduce exposure to adventitious viruses
only one raw material in the booth at a time
Weighing materials such as scoops, pipette tips, spatulas, weighing boats, and graduated cylinders are sterile and discarded or properly cleaned after use
Access to rooms monitored electronically and gowning required for technicians
gown suit, two layers of gloves, face masks, sleeve covers,

8. Air Quality & Flow
Air cleanliness classification – Class 100,000 (FDA Class D) clean room
HVAC system maintains the room at a positive pressure with respect to surrounding rooms and corridors
Room pressures continuously monitored by building automation system which collects data to show that room is maintained in controlled state
Circulate 90% of airflow through pre-filter and HEPA filter bank
HEPA filters certified and calibrated periodically (generally every 6 months)

9. Dispensing Booths

10. CIP/SIP Systems CIP (Clean In Place)
automatic cleaning of processing equipment, vessels, piping and in-line devices
minimal manual setup or shutdown
little or no operator intervention during cleaning
strong base, rinse, strong acid, final rinse with WFI
conductivity tests to monitor content of cleaning solutions and rinse water
SIP (Steam In Place)
equipment and vessels sterilized with clean steam
After sterilization system must remain pressurized to maintain sterility
Vessels used during process and all associated piping/hoses must be free of any foreign substances prior to use
include cell debris, media, cleaning chemicals, and target protein from prior batch

11. Cell Culture Media
Provides all the nutrition cells need within a narrow window of environmental conditions for optimal expression of the target protein
Major media components
Carbohydrate energy source – glucose
Nitrogen source such as amino acids
Lipids – often in the form of fatty acid
Cells also require
Trace minerals in the form of electrolytes (salts)
fetal bovine serum supplements – not so common due to risk of animal viruses
Chemically defined, serum free media commonly used – reduces threat of adventitious animal virus contaminants
Selective agents that cells require for optimal expression of the target protein
Cells with transfected gene will be able to thrive and make the target protein; cells without will die

12. Media Preparation Usually done in tanks Parameters monitored
Powdered media dissolved in high purity grade Water For Injection (WFI)
Batch Record followed with every step
Proper mixing time essential – media must be homogenous before introducing cells
Parameters monitored
pH- measures degree of acidity or alkalinity
Conductivity – measure of ions dissociated in the solution; purity of solutions used in media prep
Glucose - indicator of cell growth; main energy source for cells in growth phase
Osmolality - measure of osmotic pressure, dependent on salt concentration of media
Osmolality – the number of osmoles of solute particle per kilogram of pure solvent

13. Cell Culture/Fermentation
Cell Growth - 4 distinct phases
lag phase – cells adapt to the environment
log phase – exponential growth
plateau phase – growth rate slows- rate of proliferation equals rate of cell growth
death phase – rate of cell death exceeds proliferation rate and cells start to die
Generation – doubling of cell concentration from the original seeding cell concentration
Doubling time vary depending on the cell
E.Coli - doubling time 20 minutes – culture batch ~ 24 hours
CHO - doubling time hours – culture batch ~ 6-20 days
Goal of upstream processing is to grow cells which will produce a desired protein that will be further purified during the downstream processing steps.
Number of stages within in upstream processing – inoculum, bioreactor stage (seed and production) and primary recovery
*Have students come up with phases of growth curve

14. Cell Culture Growth Phases

15. Master Cell Bank (MCB)
Cells used for production culture batches are established from a single clone and stored frozen in banks called Master Cell Banks
stored at low temperatures in vapor phase of liquid nitrogen
(- 196°C)
suspended in cryoprotecant (DMSO or glycerol) to prevent damage to cell membrane by ice crystal formation
Preserves characteristics of original cell line
Prevents contamination and deterioration
Produced in accordance with regulatory standards (21CFR 610)

16. MCB- Cell Line Characterization
Cell line have to be stringently tested to
Confirm identity of expression construct (species identity)
Confirm purity (contamination-sterility tests for bacteria, mycoplasma, adventitious viruses)
Confirm genetic stability (coding region)
Quality assurance must be established from MCB to end-of-production/post production cells (EPC/PPC)

17. Upstream Processing Stages
Inoculum
Frozen vials of cells from cell bank thawed and added ( “inoculated”) to spinner flasks ( 100 – 500 mL) or cell culture bags ( mL)
Cell culture expanded to meet cell density and volume requirements to inoculate larger volume bioreactor
Bioreactor Seed
Cell cultures from spinner flasks or culture bags transferred “seeded” into larger volume bioreactors ( up to 20,000L)
Bioreactors are either stainless steel or disposable
Cells grow to high densities producing API in the culture media
Primary Recovery ( Harvest)
main purpose is to separate the cells from the media containing the API
Centrifugation to separate cells from media
Filtration to remove large debris

18. Inoculum
Ampoule of frozen cells released from Master Cell Bank and thawed - Out of Freeze (OOF)
Cells added to prepared media in spinner flasks or culture bags and cultured in temperature controlled (35-38°C) humidified, incubator infused with CO2 (5%)
Specific conditions to promote multiplication of the cells without producing the protein
Monitor cell concentration, viability, and pH of culture medium
Replenish nutrients when culture reaches % of it’s peak growth – ”subculture or passage”

19. Spinner flask containing cells in suspension in a biosafety cabinet

20. Culture Parameters- pH
Critical process parameter – fluctuations in pH can affect growth
Optimal pH between – 6.8 and 7.4
Incubator
Culture media contains bicarbonate which, when combined with CO2 infused into the culture incubator makes a buffering system to control pH
Bioreactor
pH measured by inline probe and bioreactor control system
pH of media decreases during cell growth as cells metabolize glucose in the presence of oxygen and produce C02
Alkaline solutions (sodium bicarbonate, sodium hydroxide) added to increase and control pH

21. Other Culture Parameters
Temperature - optimal - 37°C
C02 incubator-typically controlled either by a water bath that circulates through the walls of the cabinet (water jacketed C02) or by electric coils that give off radiant heat
bioreactor- monitored by temperature probe and Temperature Control Module (TCM)
Dissolved oxygen (bioreactors)
controlled by DO probe and computer system
based on the rate at which oxygen molecules diffuse a membrane covering a set of electrodes
low solubility in culture media

22. Maintaining & Monitoring Culture
Cell growth and viability monitored during culture by counting cells methods
Cell counting:
tallies the number of viable (living) and non-viable (dead) cells
calculate the % viability (viable/viable + nonviable)*100
calculate viable cell concentration
Accurate and consistent cell counts essential to robust production process
Viable cell concentration & % viability used as Forward Processing Criteria (FPC) & Critical Process Parameter (CPP)
Can be determined offline or inline

23. Offline Counting Methods
Trypan Blue Exclusion Method
Sample of culture must be taken from the culture vessel ( offline) to count
Live cells with intact cell membranes do not allow the trypan blue dye into the cell (remain clear)
Trypan blue enters dead cells; become blue
Time sensitive – cells must be counted as quickly as possible after staining
Used in conjunction with hemacytometer
Most commonly used cell counting method
Automated cell counting – use image analysis to automate Trypan Blue Exclusion Method

24. Inline Counting Methods
Cells are counted in the bioreactor and decreases the risk of contamination that occur during sampling
Biomass sensors
measures only viable cells
Viable cells with intact plasma membranes have a different capacitance (ability to store charge) than cells with disrupted plasma membranes (non-viable cells)
When an electric field is applied to the culture, biomass probes can measure capacitance which is directly related to concentration
Packed Cell Volume (PCV)
Subjective and does not differentiate between viable and non-viable calls
Generally used to determine cell density for cell harvest

25. Hemocytometer showing viable & non-viable cells with trypan blue stain

26. What is a Bioreactor?
Vessels or containers designed to support the optimal growth and metabolic activity of cells producing a product of interest
Can be classified in several ways including:
Type of mixing
Stirred Tank
Airlift
Mode of operation
Batch culture
Fed batch
Perfusion
Type of vessel
Stainless steel
Single use/ disposable

27. Bioreactor- Mixing Types
Stirred Tank
required gases (e.g. 02) nutrient media, cells are continuously stirred by agitator impellor (stirrer) at the bottom or top of the vessels
Baffles in the center of vessels ensures proper mixing and prevents formation of vortexes that might shear cells
Airlift
gas is pumped from below through a sparge tube within the bioreactor creating bubbles which mixes the contents of the vessels
contains baffle that guides gas up through bioreactor on one side of the baffle and then over and down the other side

28. Bioreactor –Modes of Production
Batch culture
culture grows without additions until harvest
approximately 5-8 days culture
Fed batch
depleted and/or limited nutrients are added back to the reactor
10-20 days
Perfusion (Continuous)
equal volumes of fresh media are added and culture fluid is removed from the bioreactor
can last for months

29. Bioreactor Types – Stainless Steel
Made of durable material that can accommodate high volumes (up to 20,000L) of culture
Double walled, glycol jacketed with 4 layers that provide insulation/temperature control and sterile contact for cell cultures
Very large industrial sized reactors bioreactors have fixed vessel configurations with predefined port assemblies that can not be easily reconfigured
Expensive and time consuming cleaning procedures
high costs to produce purified water and steam for cleaning (CIP/SIP)

30. Types of Bioreactors- Glass and Stainless Steel

31. Bioreactor Types – Single Use
Disposable bioreactors- intended for one time use
Components are typically made of plastic and are disposed of after use
Generally used for mammalian cultures
Cultivation chamber is inflated plastic bag
Single use technology in biomanufacturing is becoming widespread - has advantages and disadvantages

32. Advantages of Single Use Technology
Reduction or elimination of cleaning, sanitization, and sterilization steps. This reduces:
consumption of water – pure water is extraordinarily expensive to produce
energy used to produce purified water
consumption of cleaning and sanitizing chemicals (CIP)
eliminates the need to sterilize bioreactors (the vendor has done this)
eliminates the need to generate clean steam
need for cleaning and sterilization validation (and moves it to the vendor) – eases regulatory compliance

33. Single Use Advantages Continued
lower upfront capital costs – this becomes a major advantage for a small company or a new company wanting to start production quickly
faster cycle times and faster, less expensive changeover between campaigns – less fear of cross-contamination (by eliminating the need for cleaning)
lower risk – lower probability of cross-contamination with another product or microbial contamination

34. Single Use Disadvantages
Scale Limitations (only up to 2000 liter cell culture)
increasing product titers and cell densities are making this less important
Limited to mammalian cell culture - low oxygen transfer coefficient rates excludes use of bacteria
Increased reliance on outside vendors – potential supply chain problems
Concerns over leachables/extractables from the plastics
Additional consumables cost
Environmental impact – increase in solid waste that currently goes to landfill

35. Disposable wave bioreactor and its mechanics

36. Single Use Bioreactors

37. Cell Harvest
Main objective is to separate the cells from the media containing the target API
During culture samples taken at pre-determined intervals to monitor progress
Time to end culture and begin harvest depends on cell line and is pre-determined
based on the quality and quantity of the product accumulated in the bioreactor
generally highest amount of quality product is reached when cell number drops
Bacterial fermentation – cells must be lysed prior to centrifugation to free the protein contained within the cell

38. Cell Harvest Continued
2 steps:
1. Centrifugation
separate cells from culture media
Rapid spinning of the culture from bioreactor; cells sink to bottom of centrifuge
For microbial harvest lysing step prior to centrifugation to release protein product contained inside cell wall
2. Filtration – remove large debris
Depth filtration – API passes through as the filtrate along with other proteins and cell particles
Sterile grade membrane filtration – remove smaller particles and potential microbial contamination
filters with 0.22 micron pore size
Bacterial fermentation – cells must be lysed prior to centrifugation to free the protein contained within the cell

39. Cell Harvest Centrifuge

40. Types of Harvest Filters