1. Protein Pharmaceuticals (V) “Production Consideration”*
07/16/96
Protein Pharmaceuticals (V) “Production Consideration”
Dr. Aws Alshamsan
Department of Pharmaceutics
Office: AA87
Tel: *

2. Objectives of this lecture
By the end of this lecture you will be able to:
Describe the problems associated with protein formulations
Numerate strategies to improve protein formulations
Understand the difficulty of scaling up pharmaceutical protein industry

3. Problems with proteins
Large molecules
Very hard to be synthesized chemically
Unstable:
Held by weak forces
Easily destroyed in vitro and in vivo
Hard to obtain in large quantities by extraction
Loss or denaturation of many proteins during the process
Easy to contaminate
Most proteins are given parenterally
Difficult to formulate for large scale purposes
Reproducibility is a challenge

4. Microbial Considerations
Solving the problems
Microbial Considerations
Additives
Storage
Formulation
Delivery

5. Microbial Considerations
Solving the problems
Microbial Considerations
Additives
Storage
Formulation
Delivery

6. Microbial consedirations
Sterilization:
It is impossible to sterilize the end product
All equipments must be sterilized
Assembled in aseptic conditions “BSL and Cleanroom”
Quality control:
Viral testing
Bacterial testing
Pyrogen testing

7. Cleanroom
An environment of controlled level of contamination that is specified by:
The number of particles/volume
Particles size
Air entering is HEPA filtered to exclude dust, airborne microbes, and aerosol particles
Working staff must wear personal protective equipments (PPE)

8. Laminar Flow Cleanroom
Turbulent Cleanroom
Laminar Flow Cleanroom

9. Maximum Particles/ft³
Cleanroom
US FED STD 209E cleanroom standards
Class
Maximum Particles/ft³
ISO equivalent
≥0.1 µm
≥0.2 µm
≥0.3 µm
≥0.5 µm
≥5 µm 1 35
7.5 3
0.007
ISO 3 10
350 75 30
0.07
ISO 4
100
3,500
750
300
0.7
ISO 5
1,000
35,000
7,500
3000 7
ISO 6
10,000
350,000
75,000
30,000 70
ISO 7
100,000
3.5×106
750,000
300,000
700
ISO 8
In November 2001, US FED STD 209E was cancelled

10. Cleanroom ISO 14644-1 cleanroom standards Class Maximum Particles/m³
FED STD 209E equivalent
≥0.1 µm
≥0.2 µm
≥0.3 µm
≥0.5 µm
≥1 µm
≥5 µm
ISO 1 10
2.37
1.02
0.35
0.083
0.0029
ISO 2
100
23.7
10.2
3.5
0.83
0.029
ISO 3
1,000
237
102 35
8.3
0.29
Class 1
ISO 4
10,000
2,370
1,020
352 83
2.9
Class 10
ISO 5
100,000
23,700
10,200
3,520
832 29
Class 100
ISO 6
1.0×106
237,000
102,000
35,200
8,320
293
Class 1,000
ISO 7
1.0×107
2.37×106
1,020,000
352,000
83,200
2,930
Class 10,000
ISO 8
1.0×108
2.37×107
1.02×107
3,520,000
832,000
29,300
Class 100,000
ISO 9
1.0×109
2.37×108
1.02×108
35,200,000
8,320,000
293,000
Room air

11. Viral Decontamination
There is no well-determined mean to detect viruses in the cell culture
Each lab has a level of biocontaminents AKA Biosafety Level (BSL):
BSL1:Well-characterized agents not know to cause disease to a healthy adult human being
BSL2: BSL1+ agents of moderate potential hazard to personnel and environment (e.g. HBV and Salmonella)
BSL3: Agents which may cause serious or potentially lethal disease after inhalation but to which treatment is available (e.g. TB, Anthrax, and SARS)
BSL4: High individual risk of aerosol-transmitted lab infection that cause severe or fatal diseases to which no treatment or vaccine is available (e.g. Ebola and Marburg)

12. Viral Decontamination
Viral contamination can be from the host cell line or nutrients present in the growth media (e.g. FCS)
Category
Type
Example
Inactivation
Heat
Pasteurization
Radiation
UV-light
Dehydration
Lyophilization
Cross linking
Formaldehyde
Neutralization
Antibodies
Removal
Chromatography
Affinity chromatography
Filtration
Ultrafiltration
Precipitation
Cryoprecipitation
but these processes may be harmful to the product

13. Bacterial Decontamination
Filtration sterilization of the final product by bacterial filter “0.22 mm membrane filter”
Antibiotics must be added to the cell culture to inhibit bacterial contamination
What if the expression system is bacterial?
Complete removal of antibiotic residues from the final product is very difficult

14. Pyrogens
The process of pyrogen removal AKA depyrogenation refers to the removal of pyrogens such a “endotoxins” from solutions.
Property
Exotoxin
Endotoxin
Chemistry
Secreted proteins
Shed lipopolysaccharide
Source
Gram (+ve) or Gram (-ve) bacteria
Gram (-ve) bacteria
Symptoms
Specific action on target tissue
Fever, diarrhea, vomiting, shock
Toxicity
High / Fatal
Weak / Rarely fatal
Immunogenicity
Causes neutralizing Ab production
Insufficient Ab production
Toxoid potential
After formaldehyde treatment
None
Fever potential
Rarely
Pyrogenic

15. Pyrogens
Lipopolysaccharide is a component of Gr (-ve) bacteria cell wall
Pyrogenic part

16. Pyrogen Testing Rabbit Test: LAL Test:
Rabbits have similar endotoxin tolerance to humans
Costly method and time consuming
Inability to quantify the endotoxin level
LAL Test:
Limulus Amebocyte Lysate (LAL) test
FDA-approved for in vitro pyrogen testing
High sensitivity EU/mL
Only detects LPS
Gives false positives with Glucans

17. Pyrogen Removal
Simple filtration sterilization and standard autoclaving conditions do not remove pyrogens
Dry heat for 30 min at 250 oC would breakdown the endotoxin
All equipments used in the production process must be endotoxin free
The FDA’s maximum permissible endotoxin limit is 5 EU/kg/hr
Intrathecal endotoxin limit is 0.2 EU/kg/hr
Sterile water for injection is allowed to contain EU/mL

18. Pyrogen Removal Ion Exchange Chromatography:
LPS is highly negative
Anion exchanger
Ultrafiltration and Reverse Osmosis:
LPS has high molecular weight >10kDa
Ultrafiltration
Reverse Osmosis

19. Pyrogen Inactivation
Hydrolysis in order to cleave Lipid A from the polysaccharide component, Oxidation using hydrogen peroxide, and Heating at 250 oC for 30 minutes are commonly used methods inactivate endotoxin on solid surfaces. However, these methods would harm the therapeutic protein. Therefore, it is important to work with sterile endotoxin-free equipments under aseptic condition.

20. Cellular DNA
Mammalian expression systems are immortalized cell lines by stable oncogene transfection
Recombinant products may get contaminated with oncogen-bearing DNA fragments in the final product
Purification process MUST remove cellular DNA and RNA
DNA concentration in the final product should not exceed 10 pg/dose

21. Protein Contaminants Source:
Growth media (FBS)
Host cells
Ligands from affinity chromatography columns
Host version of the protein can be co-purified with the protein of interest
Large-scale production prefers the use of serum-free media (e.g. in mAbs production) but this causes insufficient growth and lower yield of production
Foreign protein contaminants can be hazardous and immunogenic. If not purified, they lead to miss-interpretation of the produced protein’s immunogenicity profile

22. Microbial Considerations
Solving the problems
Microbial Considerations
Additives
Storage
Formulation
Delivery

23. Microbial Considerations
Solving the problems
Microbial Considerations
Additives
Storage
Formulation
Delivery

24. Additives Active ingredient Solubility enhancer
Anti-adsorption/aggregation agent
Buffer components
Preservative/anti-oxidant
Lyoprotectant/cryoprotectant
Osmotic agents
Delivery systems

25. Solubility enhancer Problem: Solution:
Aggregation and precipitation especially with non-glycosylated proteins
Solution:
Proper pH and ionic strength
Cationic amino acids (Lys and Arg)
Surfactants (e.g. SDS)

26. Anti-adsorption Anti-aggregation
Problem:
Hydrophobic sites causes adhesion and adsorption to solid interfaces and leads to unfolding and aggregation
Solution:
Proper pH and ionic strength
Surfactants (e.g. phospholipids and SDS)
Competitor protein (e.g. Albumin)

27. Buffer components Problem: Solution:
Protein solubility and stability depend to a great extent on the pH of the surrounding environment. Temporary change in the pH can cause aggregation
Solution:
Add buffer components
Citrate (pH 3-7), acetate (pH 3-7), and phosphate (pH 7-11) buffers
Choose buffer systems that do not crystallize during freezing

28. Preservatives and Anti-oxidants
Problem:
Oxidation occurs to (Met, Cys, Trp, Tyr, and His)
Contamination with microorganisms expecially in multiple-dosing dosage forms
Solution:
Replace oxygen in the vial with inert gas
Ascorbic acid
Preservatives at bacteriostatic concentrations (e.g. p-hydroxybenzoic acid and thimerosal “thiomersal”)

29. Osmotic Agents Problem: Solution:
Most proteins are given parenterally. Therefore, they must be administered as isotonic solutions. However, excipients used in this regard may influence protein structural stability
Solution:
Sugars (e.g. sucrose) and polyhydric alcohols i.e. sugar alcohol e.g. glycerol and PEG improves protein stability through preferential exclusion

30. Microbial Considerations
Solving the problems
Microbial Considerations
Additives
Storage
Formulation
Delivery

31. Storage Aqueous solutions: Freeze-dried form (Lyophilized)
Stability of protein solutions depends on pH, ionic strength, temperature, and stabilizers
Smooth walled glass
Air-tight container
Dark
Freeze-dried form (Lyophilized)
Dried form in compact state (pills)

32. Freeze Drying
Presence of water in the protein solution promotes chemical and physical degradation, which reduces the expected shelf life
Freeze drying removes water through sublimation and not evaporation

33. Freeze Drying

34. Freeze Drying

35. Freeze Drying Steps The freeze drying process consists of three steps:
Freezing:
Crystallization of water molecules (bound and unbound to protein/excipients)
Primary drying:
Removal of unbound water molecules by sublimation
Secondary drying
Removal of protein/excipient bound water by sublimation

36. Freeze Drying Steps
In absence of proper excipients, irreversible damage to the protein

37. Lyoprotectant/Cryoprotectant
PEG:
Coats the protein
Not a very good stabilizer
Sucrose:
Freezes the water molecules around the protein (preferential exclusion)
Also preservative above 60%
Lyoprotectants prevent over drying of proteins during freeze drying

38. You are now able to:
Describe the problems associated with protein formulations
Numerate strategies to improve protein formulations
Understand the difficulty of scaling up pharmaceutical protein industry

39. Next Lecture