1. Bio-manufacturing Overview
Hyuncheol Kim, PhD
Drug Delivery & Tissue Engineering Laboratory
Sogang University

2. Manufacturing Overview
Manufacturing Steps
- Upstream
- Recovery
- Downstream
The Rules = Compliance!!!
- FDA Guidelines
- cGMP
- Batch Records, SOPs

3. Process Flow Diagrams Process flow diagrams (PFD):
describe a specific product’s manufacturing processes
indicates the general flow of the process through a manufacturing facility
contains only important process details (i.e. temperature, pH, and cell densities) and steps
shows major equipment in the facility
PFDs are used to:
assist in the transfer of a process to manufacturing from development
write process batch records
describe the process to the FDA in regulatory documents

4. Upstream PFD Example

5. Downstream PFD Example

6. Detail of One Step Example

7. Upstream PFD Example

8. Upstream – Seed Thaw & Expansion in Shake Flasks
Purpose: To aseptically produce an inoculum of sufficient volume, cell density, and viability for inoculation of the first bioreactor
Procedure: Thaw a Cell Bank Vial & continually passage into subsequently larger flasks to increase the total cell culture volume
Maintain healthy cells
Replace the nutrients previously consumed by cells with nutrients from fresh media
Remove potentially toxic by-products of metabolism, ie. ammonia and lactic acid
Increase the total volume of cell culture in stages
This allows you to start with a cell bank vial containing 1.6-mL of cell culture and end with 20,000-L of cell culture
Usually, batch phase

9. Upstream – Seed Thaw & Expansion in Shake Flasks

10. Seed Build-up Terminology
Inoculation – The introduction of cells to the cell culture medium
Passage – The act of diluting cells in new medium from a high density to a low density
Seed Culture – The culture to passage. The culture used to inoculate a new flask
Viable Cell Density (VCD) – The total number of cells per milliliter of culture that are alive
Viability – The percentage of live cells in culture
Inoculation VCD – The density of the new culture after the seed culture is passaged (typically low)
Final VCD – The density of the seed culture prior to passaging
Split Ratio – the ratio of the volume differences between subsequent stages

11. Seed Build-up

12. How to Passage Cells
Sample the cell culture to be passaged aseptically
Make sure the culture is well mixed before removing a sample
Perform a cell count of seed culture on an automated cell counter machine (ViCell)
Using the following equation to determine the volume of seed cell culture needed to inoculate a new flask.
C1 x V1 = C2 x V2
C1 = Cell Density of Seed Culture
V1 = Volume of Seed Culture Needed (Unknown)
C2 = Inoculation Cell Density of New Culture
V2 = Volume of New Culture

13. Seed Build-up Controls
Incubator
Agitation (oxygenation)
CO2 (help aid in pH stabilization)
Temperature
Humidity
Bioreactors
Agitation
pH control
DO control

14. Bioreactors / Shake Flasks
It is best to transfer the cell culture into a bioreactor as soon as possible because cells grow better in a bioreactor than they do in a shake flask.
Advantages to seed build-up in bioreactors:
Provide a controlled environment
Easier to maintain sterility
No hood work in a biological safety cabinet required
Build-up cells to higher volumes
Less likely chance for contamination
Disadvantages to seed build-up in bioreactors:
Cost of operation is greater
Requires longer time to set up (CIP, SIP, etc)
Some companies start the in vessels as small as a 5 L bioreactor

15. Designing a See Build-up Process
When designing a seed build-up process, it is desired to make it as short as possible.
It is not desired for the cells to produce protein, thus the goal is to quickly grow the culture to the necessary volume and cell density required to inoculate the production bioreactor.
In order to design a seed build-up process, the following information is required:
The maximum VCD the cells can grow to in the batch media.
The time required for the culture to reach the maximum VCD.
The minimum VCD allowed for inoculation. Cells like to be near other cells; if the inoculation VCD is too low, the culture will not grow.
The maximum split ratio the culture can withstand.
You also need to know the following about the facility’s bioreactors / equipment:
The largest size shake flask, spinner flask, or roller bottle that is available
The working volume of the seed build-up bioreactors.
The working volume of the production bioreactor.
The minimum and maximum working volumes in each bioreactor.
Once you have all the information, the optimal process can be designed!

16. Batch Cell Growth

17. Upstream PFD Example

18. Production Bioreactor
Purpose: to produce the desired product!
Production bioreactors can be: Batch, Fed-batch, or Perfusion
Mammalian processes are on average 7 – 14 days

19. Upstream PFD Example

20. Recovery Purpose First stage of purification
Remove the final product from the cellular material
Most products are extracellular
Remove cells from the liquid containing the product
Some products are intracellular
First must break the cell membrane to get the product into solution (mechanical presses, detergents)
Next remove the cell debris

21. Recovery
Consists of a series of steps ending with a final sterile filtration of the material
Common methods include
Centrifugation
Homogenization / Microfluidization
Depth Filtration
Microfiltration
Tangential Flow Filtration
Process is designed based on the type of starting culture
Cell density
Cell type

22. Downstream PFD example

23. Purification
A series of filtration and chromatography steps designed to purify the product while maintaining its bioactivity
Need to remove viruses, host cell proteins, and exchange the buffer in order to get the product into the formulation buffer
Purification steps are not sterile processes, however sterile filtration between steps is required
Also, require viral filtration – utilizes very small micro filters; expensive

24. Downstream PFD Example

25. Types of Chromatography
Flow-Through Mode
Is when the target molecule does not bind to the column
So the resin is selected to capture contaminants rather than the target protein
Capture Mode
Is when the target molecule binds to the column.
The contaminants may either flow through or bind to the column
So the resin is selected to capture the target protein

26. Stationary phase (resin)
Mobile phase (buffer)
Mobile phase
Molecules spend different amounts
of time between the two phases
When a mixture of molecules is loaded on to a column;
Molecules that spend 100% of the time in the stationary phase are “bound”
Molecules that remain 100% in the mobile phase are in the “flow through”

27. Protein Capture

28. Step Gradient/ Step Elution:
Gradient Elution: Modify solute interaction with the stationary phase by changing the composition of the mobile phase.
Step Gradient/ Step Elution:
Change mobile phase composition in a stepwise or instantaneous manner
time
Linear Gradient:
Change mobile phase composition in a continuous (linear) manner

29. Chromatography Terms Equilibration
When buffer is pumped though the column to adjust the pH and conductivity in preparation for loading the protein
Load
This is the step where the protein solution is pumped into the chromatography column.
Wash
The wash step can have several purposes, it can be used to finish pushing the load material though the column, it can be used to remove contaminates from the column or it can be used to begin separating molecules on the column prior to elution
Elution
This step is where the pH and conductivity of the buffer are adjusted so that the target protein migrates to the mobile phase and flows out of the column into a collection container

30. Chromatography Terms Strip / Regeneration
A solution that is flowed though the column to remove remaining protein / contaminants and return the column to its original state
Cycle
1 cycle is when the series of steps (Equilibration, load, wash, elution….) is completed one time. Multiple cycles per batch may be used on the column as needed. Cleaning is done when the batch is finished and not between each cycle.
Cleaning / Sanitization
This step is used to remove any remaining contaminates on the column and to eliminate any microbes that may have entered the column
Storage
After cleaning, a storage solution is pumped into the column to ensure the column resin is maintained in stable conditions until the next run. Storage solution is usually bacteriostatic (inhibits bacterial growth)

31. Chromatography Diagram

32. Downstream PFD Example

33. Bulk Product Fill Bulk = Product in bags
Bulk product is the final product from the purification step (prior to filling)
This product goes on to be filled into syringes / vials, lyophilized and either placed in vials or pressed into tablets
Bulk = Product in bags

34. FDA Compliance

35. Compliance
Compliance - Act according to the standards set by the regulations and guidelines

36. There are three GXPs: 1. Current Good Manufacturing Practices (CGMP):
21 CFR Part 210 & 211
2. Good Clinical Practices (GCP):
Law that governs the action and environment of those working in clinical testing of drugs and medical devices on human beings.
21 CFR Parts 56, 312, and 314
3. Good Laboratory Practices (GLP):
Enforced code of practice to reduce accidents affecting research projects or manufactured products.
21 CFR Part 58

37. Overall Purpose of cGMPs
Establish Industry Standards
Insure Traceability
Insure Accountability
Promote Uniformity
Install Quality
It’s the Law!
Title 21 of U.S. Code of Federal Regulations - “21 CFR”
...To assure that all pharmaceutical, biologic, diagnostic and medical device or drug products meet all the requirements of the Federal Food, Drug and Cosmetic Act as to safety, and have the identity and strength and meet the quality and purity characteristics which they purport to have...
Parts 210.1, 210.2

38. How do Companies Comply
Standard operating procedures (SOPs) must be followed
Manufacturing Processes must be validated
Equipment must be validated
Staff must be properly trained

39. Standard Operating Procedures
Used for routine steps, equipment setup, procedures, tests
Ensure that the procedure is performed the same way every single time, no matter who performs it
People must be trained on the SOP
Examples – taking a pH measurement; installing filters on the bioreactor; operation of the chromatography skid

40. SOP

41. Batch Records
Used to record all the data coming from on batch for a process
Batch records would be created for each step
Raw data collected about the process
Entries must be verified by additional person
Examples – The Production Bioreactor for Protein X
Collect the daily cell counts
Mention if you needed to change the pH or agitation

42. Batch Records

43. Similarity and Difference between SOPs and BRs
Version tracked to keep track of changes
Reviewed and approved by multiple people / depts
Batch Records are issued for a particular batch
Given an assigned number for the document
Only used for a particular step for a particular product
Version tracked to keep track of changes
Reviewed and approved by multiple people / depts
Used across products and production sites
Stored in the lab as a reference

44. In the compliance world documentation is KEY
If you didn’t write it down it never happened!!

45. Thank You