1. Chapter 11 -Downstream Processing
Northeast Biomanufacturing Center and Collaborative

2. Downstream Processing Goals
The overall goal of the downstream purification process is to produce a drug with required purity, efficacy, and reasonable cost per unit
Starts with harvest from bioreactor and finishes with a highly purified and appropriately concentrated product ready for final formulation and packaging
3 stages
Capture or Recovery – separation of the product of interest from the cells of the bioreactor
Intermediate Purification – removal of bulk contaminants (host cell proteins, adventitious viruses, traces of DNA, endotoxin)
Polishing – elimination of trace contaminants and impurities (inactive or unwanted isoforms of the target molecule, common impurities)
Chapter uses monoclonal Ab production as the example
Capture (on board)
removal of all micro-particulates and colloidal materials
removal of the majority of water, growth medium supplements and small molecule solutes
isolation of product from proteolytic enzymes or other sources of degradative elements from the cell culture harvest

3. Downstream Processing in Biopharmaceutical Manufacturing
Harvest by Centrifugation
Clarification by Depth Filtration
Sterile Filtration (MF)
Tangential Flow Filtration (UF/DF)
Low Pressure Liquid Column Chromatography

4. Downstream Processing and Purification

5. Process Filtration Used at several stages of the downstream process
Use membrane filters to separate the protein of interest from other proteins and molecules in the sterile filtered, clarified medium.
3 General Types
Microfiltration – used at the start of the downstream processing
Ultrafiltration – used between chromatography steps to concentrate the products and change the buffer conditions (diafiltration)
Sterilizing grade direct flow filtration – eliminates microbial organisms and insoluble proteins, removes adventitious and endogenous viruses, and sterile filters the product for final formulation

6. Process Filtration- Flow Types
Two general flow types:
Direct Flow Filtration (DFF) – process fluid crosses the membrane in a perpendicular flow direction
provides little or no prevention of particulate build up or concentration of other elements that do not fit through the pore structure
Often used for particulate removal, sterile filtration, and virus removal/clearance filtration
Tangential Flow Filtration (TFF) –flow sweeps across the active filtration surface
minimizes pore plugging and surface fouling

7. Direct Flow Filtration
Random
Internal plugging
Dramatic flux decline over time

8. Tangential Flow Filtration
No internal plugging
Low pressure resistance
High flux performance

9. Schematic of TFFU Operation

10. Direct Filtration Systems

11. Tangential Flow Filtration Units

12. Downstream Processing – Liquid Chromatography
Separation techniques based on the differential interaction of molecules between a stationary and a mobile phases
In Greek "Chroma" means "color“ and "Graphein" means "to write”
Some constituents bind or interact with the stationary phase (packed resin made up with immobile matrix such as cellulose or agarose, synthetic polymers and silica) while others pass through the stationary phase with no interaction
Molecules move past stationary phase in different rates based on their difference in their relative attraction to the mobile phase
The bound or interacting components are then removed or eluted by the gradual or step-wise change of the composition of the mobile phase (liquid buffer or gas that moves pass the stationary phase).

13. Chromatography System Components
Columns containing stationary phase
Mobile phase pumps
Enable a wide selection of process fluids and/or proportional binary mixing to adjust conditions of conductivity and pH
Series of pre- and post-column sensors
UV-Detects proteins coming out of the column by measuring absorbance at 280nm
conductivity measures the amount of salt in the buffers coming out of the columns – high salt or low salt are often used to elute the protein of interest from the chromatography beads
pH- measures the pH during binding and elution
flow rate – monitors rate of flow of mobile phase pumps

14. Components of a Liquid Chromatography System

15. Types of Chromatography
Bind-elute methods – product binds to resin and bound components removed (eluted) by the gradual or step-wise change of the composition of the mobile phase (buffers)
Flow-Through methods – product passes through while unwanted elements are bound to resin

16. Chromatography-Bind Elute Mode

17. Typical Chromatogram

18. Liquid Column Chromatography Process
PURGE air from Column use Equilibration Buffer
PACK column with Beads (e.g. ion exchange, HIC, affinity or gel filtration beads/media
EQUILIBRATE column with Equilibration Buffer
LOAD column with Protein of Interest in Equilibration Buffer
WASH column with Equilibration Buffer
ELUTE protein of Interest with Elution Buffer of High or Low Salt or pH
REGENERATE column or Clean and Store (NaOH)
The processes in red will be carried out today (Day 3) in small plastic columns packed with beads

19. Key characteristics of packed bed resin system

20. Chromatography- Stationary Phases
Stationary phase (resin) functional groups
Charged
Specific biochemical make-up
Hydrophobic
Combination of above (mixed mode)

21. Types of Column Media/ Resins
Hydrophobic Interaction (HIC)
Ion Exchange (anion- AEX and cation exchange -CEX)
Affinity
Gel Filtration (Size Exclusion)

22. Column Qualification
When packing columns, flow of the resin and pressure should be controlled and monitored
After column is packed, integrity and efficiency of column bed should be determined
Efficiency calculated from number of theoretical plates (N) and height equivalent of theoretical plates (HETP)
HETP normalizes the column of the column to length of the column

23. Column Efficiency
To calculate the number of theoretical plates use the equations below:

24. Validation /Qualification of Solvents and Media
Sterilizing grade filter compatibility studies on filtration devices
Spike a sample of the drug product with bacteria or bacterial spores at various stages in the process to demonstrate microbial clearance as well as the maintenance of the drugs’ chemical stability and biological activity
Detection and quantification of leaching ligand during affinity chromatography to prove the drug substance is free of leaching ligand such as protein A
Validation of contaminant removal
spike and track a known and excessive quantity of the contaminant through the downstream process

25. Viral Clearance and Inactivation
According to FDA, viral clearance on the final product will not only be from direct testing for their presence, but also from demonstration that the purification regimen is capable of removing and inactivating the viruses
For each downstream processing step, the effectiveness of viral removal or inactivation must be validated using virus challenge or spiking studies where model viruses are added at known high titers than tracked through each step of the process typically via infectivity assay
For mAb purification processes, a low pH exposure at pH such as the elution buffer of the protein A column combined with a viral filtration is accepted by regulatory agencies as a virus clearance method.