1. Upstream Processing Overview
miniBIOMAN 2017

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- Active Pharmaceutical Ingredient eg growth factor, enzyme, antibody
Produced by mammalian cell culture (Chinese Hamster Ovary - CHO cells, mouse myeloma, NSO cells), or bacterial or yeast culture
Mammalian cells
can modify proteins (post translation modifications required for Ab activity)
harvest can be made without cell lysis
CHO cells most commonly used
Bacterial cells
do not usually have the machinery to secrete the desired product into the media
cells must be lysed during harvest

4. Biomanufacturing Process Flow – Upstream Processing

5. Upstream Processing Overview
Product formation
Downstream
Product purification
Quality Control
Product safety and efficacy

6. Production Process Flow Diagram

7. Upstream Processing - Cells
Cells used in upstream processing:
contain the transfected gene (s) 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

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

9. Air Quality & Flow Clean rooms
Air cleanliness classification – Class 100,000 (FDA Class D) clean room, class 100 biological safety cabinet for initial inoculation
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)

10. 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 – no longer 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

11. Cell Culture 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 type
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

12. Cell Culture Growth Phases

13. Monitoring cell growth
Parameters monitored
pH- measures degree of acidity or alkalinity
Cell growth – viable cells/ml
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
Lactate byproduct of glucose metabolism
Optical density – monitors growth
Osmolality – the number of osmoles of solute particle per kilogram of pure solvent

14. 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

15. Inoculum stage

16. Production Strategy
Batch: the culture generally goes through the growth cycle of lag, exponential, plateau, and death; the production cycle is short in this type of strategy; protein is harvested one time at the end of the production cycle
Fed batch: the culture goes through the growth cycle of lag and exponential phases; additional feed (glucose and other nutrients) is added before the cells reach plateau phase, allowing the cells to continue growing and producing protein; eventually the cells will reach the plateau and death phase, completing the production cycle; protein is harvested one time at the end of the production cycle.
Perfusion: the continuous growth of cells within a determined amount of time, with continuous harvest of product during cell growth achieved by a liquid-solids (cell) separation device within or adjacent to the production bioreactor; multiple harvests of protein are obtained from the bioreactor.

17. Manufacture of Adcetris
Upstream processing
Downstream processing

18. Manufacture of Adcetris

19. Production bioreactor adcetris
Cells in upstream process have been through intense study for many years

20. Emerging trends in upstream processing
A. Single use technology
Single use manufacturing technologies, like wave bags and platforms for scale-up
2,000L max disposables for production
Sometimes 6 x 2000L bioreactors for production
Modular construction of facility that can be assembled together rapidly

21. Emerging trends in upstream processing
B. Increase in cell titers
mAbs can be expressed at titers of >5g/L in a 14 day fed-batch production
Due to advances in :
Cell line expression vectors
Clone selection
Cell culture medium
This allows the use of smaller scale bioreactors for production runs

22. Emerging trends in upstream processing
C. Continuous production

23. From Traditional to Integrated Continuous Bioprocessing

25. 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)

26. Cell counting
Automated cell counting: use image analysis to automate the Trypan Blue exclusion method, Examples include Vi-Cell, Cedex, and Nova units.
Trypan Blue/ Dye Exclusion Method. : trypan blue passes through the leaky membrane of a dead cell. Hence dead cells exhibit a distinctive blue color under a microscope. Since live cells are excluded from staining, this staining method is also described as a Dye Exclusion Method.
-This method is time sensitive, and the cells must be counted as quickly as possible after staining. The Trypan Blue will begin to kill the cells over time. This method is among the most commonly used cell counting methods. For manual counting, these are used in conjunction with a manual hemocytometer
Biomass sensor: these sensors operate based on cells with intact plasma membranes (living, viable) having different capacitance (ability to store a charge) than dead cells with disrupted plasma membranes. The cells are put through an electric field; and the capacitance can be directly related to cell concentration.

27. Manual counting

28. CHO DP12 anti IL-8 Mab

29. Monoclonal Antibody Therapeutics
More than 50 mAbs have been approved and sales of mAbs are expected to reach $125 billion by 2020, with over 300 mAbs currently in clinical development. Explosive growth of this class of biopharmaceutical in the last 20 years due to:
ability to bind to specific targets with high specificity and affinity
ease of developing human or humanized sequences to a target
mAbs have been approved for a wide range of indications covering oncology and auto- immune disorders and rare disease indications

30. Monoclonal antibody therapeutics

31. Cell line
CHO DP-12 clone#1933 [CHO DP-12, clone#1933 aIL8.92 NB 28605/14]ATCC® CRL-12445™

32. CHO DP 12
The clone#1933 cell line was derived by co-transfecting the CHO cell line DP-12 using Lipofection with the vector p6G4V11N35A.choSD.9 designed to coexpress variable light and heavy regions of the murine 6G4.2.5 monoclonal antibody. Clones were selected in methotrexate.

33. CHO DP12 cells
The cells are reported to produce 150 mg/L recombinant human anti-IL-8.  
Antibodies inhibit IL-8 binding to human neutrophils. They also show equivalent neutralizing capabilities to inhibiting IL-8 mediated human neutrophil chemotaxis
Therapeutic anti IL-8 mAb
HuMax®-IL8 is a high affinity fully human antibody directed towards IL-8. HuMax-IL8 is in development for the treatment of solid tumors under an agreement with Bristol-Myers Squibb. An IND for a Phase Ib study of HuMax-IL8 for the treatment of metastatic solid tumors was filed in June 2015.

34. CHO DP12 Growth requirements
DMEM, high glucose
10% superlow IgG serum
(or FBS if stopping after upstream)
Insulin/transferrin
methotrexate

35. IL-8
IL-8 is a small 72 amino acid peptide
Molecular weight 7.8 kDa

36. CHO DP12 Upstream Processing
Inoculum
- 1ml from cell bank to inoculate 100ml spinner flask
Grow for 5 days
Monitor growth
Scale-up
Inoculate bioreactor
100ml to 1L medium
Harvest
Centrifuge cell culture
filter

37. 3 week experiment in Biomanufacturing course
2/7 & 2/9
Quality Assurance (Chap7)
Guest speaker
Chapter 7
Popcorn GMP activity
2/14 & 2/16
Quality Assurance
Upstream Processing
Chapter 10
FDA group activitiy
2/21 & 2/23
Upstream Processing CHO-DP12 batch culture production of recombinant anti IL-8 mAb
Inoculation, spinner flask culture, sampling and QC
Batch Culture SOP and Batch Record
Spinner flasks media hold
Inoculate spinner flasks (day 0-day 4 testing)
Bioreactor Sterilization, pH probe calibration and installation
2/28 & 3/2
Bioreactor operation, inoculation
Bioreactor Operation SOP 
Applikon bioreactor operation,
spinner flask day 6 testing
prepare bioreactor-set temp, stirrer, DO control
inoculate bioreactor and day 0-day 3 testing
3/7 & 3/9
Bioreactor harvest
Chapter 10 and chapter 11
TFF SOP
bioreactor harvest, CIP, centrifugation, filtration and TFF
3/14 & 3/16
No Class
SPRING BREAK
ENJOY!