1. Manufacturing process of biological products: drug substance : Filtration, Dialysis Inactivation
Wisit Tangkeangsirisin, PhD.
Biopharmacy Department
Faculty of Pharmacy
Silpakorn University
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2. Lecture Outlines Introduction Conclusion
Recombinant Proteins significance in Pharmacy
Feature of Recombinant Protein
Upstream Process technology
Filtration/Concentration
Inactivation
Conclusion
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3. Type of Vaccines
live, attenuated bacteria (e.g. bacillus Calmette–Guérin (BCG), used to immunize against tuberculosis);
dead or inactivated bacteria (e.g. cholera and pertussis vaccines);
live attenuated viruses (e.g. measles, mumps and yellow fever viral vaccines);
inactivated viruses (hepatitis A and polio (Salk) viral vaccines);
toxoids (e.g. diphtheria and tetanus vaccines);
pathogen-derived antigens (e.g. hepatitis B, meningococcal, pneumococcal and Haemophilus influenzae vaccines).

4. Bacterial Vaccines Attenuated, bacteria dead or inactivated bacteria
BCG  could not form tuberculosis
dead or inactivated bacteria
heat treatment;
treatment with formaldehyde or acetone;
treatment with phenol or phenol and heat;
treatment with propiolactone.

5. Inactivated Vaccines
inactivated or killed organisms by chemical or physical means
TRENDS in Biotechnology Vol.23 No.2 February 2005

6. Pathogen-derived antigens
Traditional Antigen based vaccine
Surface antigen
Toxoids (Inactivated toxins)
Recombinant vaccines (subunit vaccine)

7. Production of Vaccines
Attenuated vaccines
Attenuation (natural/engineered/growth control)
Bacterial or viral expansion
Killed / Inactivated vaccines
Killed or inactivation (heat/chemicals)
Surface antigen vaccine
Bacterial/viral expansion
Antigen extraction
Recombinant vaccine
Engineer into suitable expression system
Cell expansion  antigen extraction

8. Vaccine Manufacturing Strategies (Killed Vaccines, Toxoids)
Pathogen Seed
wP, HAV
Culture
Inactivation
Vaccine
Rabies Flu
Purification
Vaccine
Antigen purification
aP, D, T,
Split IIV
Inactivation
Vaccine

9. Formalin Most widely used First developed for IPV, then HAV
Now for influenza and others (JEV, TBE)
37 % w/v formaldehyde (13.3 M) equals 100 % formalin and
1/4,000 dilution of formalin is thus identical to % formaldehyde (i.e., 3.3 mM or 100 μg/ml formaldehyde)
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10. Inactivation by Formalin in Flavivirus vaccine
JE and TBE
No specify inactivation condition from WHO or Ph Eur
Examples are available (JEV days at 4C, 1:2000)
For WHO approval, testing for completeness of inactivation requires testing of 25 human doses for JEV (WHO 2007b) and 20 doses for TBE (WHO 1999), while the Ph. Eur. requires a minimum of 10 human doses for TBE (Ph. Eur. 2011f).
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11. Inactivation by Formalin in Piconavirus vaccine
WHO Require linear regression of viral titre (3x) at 37C
Extrapolation of inactivation kinetics curves
Bulk must be tested for completeness of inactivation
Inactivation kinetics of poliovirus at 36–37 °C according to Salk’s first-order hypothesis
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12. Formaline Inactivation
Methods for formaldehyde inactivation vary greatly between vaccines. Differences
lie in formalin concentrations (from 0.08 to % w/v), time of inactivation
(from days to months), and temperature (usually 4 or 37 °C)
Now in IIV
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13. Beta-propiolactone (BPL) Inactivation
Used in influenza and rabies vaccine
Unstable in aqueous (rapid hydrolysis)
Completely elimination at 2h 37C (advantage over formaldehyde, no requirement to test residual)
Direct interaction with nucleic acid
generally set at 4 °C for 18–24 h with a BPL concentration of 0.1–0.25 %,
this can differ per pathogen
Require shorter time and lower temperature to inactivation (vs. formalin)
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14. Overview of FDA approved inactivate influenza vaccines
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15. Other inactivation methods in development
Hydrogen peroxide
Zinc finger reactive compounds (in RSV vaccine)
Gamma radiation
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16. Inactivation
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18. Filtration
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19. Filtration
A mechanical or physical operation which is used for separation of solids from liquids
Two main types of Filter Medias:
Surface Filtration (Membrane Filtration) (eg. Buchner Funnel, Cross Flow Filter)
Depth Filtration (eg. Sand Filter)
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21. Filtration: Classification
Surface Filtration
Depth Filtration
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22. Parameter Surface Filters Depth Filters
Deformable Particles
May blind off pleats
Recommended - adsorptive retention
Non deformable Particles
Removes narrow range
Removes broader range of particles
Rating
Absolute or nominal
Classification/Clarification
Classification
Clarification
Economic - Particle Retention < 10 Micron
Holds more dirt than depth, handles higher flow rate
More economical than pleated at greater than 10 microns
Cartridge Cost *
More expensive initially than depth, fewer replacements, holds more dirt
More economical initially than pleated, holds less dirt
Housing Cost *
Fewer cartridges - smaller housing
More cartridges-bigger housing
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23. Filtration Techniques
Dead-End Filtration
Crossflow Filtration
All fluid passes through membrane
Larger Particles stop on the membrane
Form “Filter Cake”
Batch operation
Fluid feed stream run tangential to the membrane
Some particles stop, other flow across membrane
Prevent “Filter Cake”
Continuously operation
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25. Cell Isolation/Harvesting
Cells
Cell
Concentrate
Membrane
Cell
Suspension
Cell-free culture
medium
Cell-free culture
medium
Dead End
Filtration
Cross Flow Filtration
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30. Ultrafiltration/Diafiltration
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31. Ultrafiltration
a variety of membrane filtration in which hydrostatic pressure forces a liquid against a semipermeable membrane. 
Suspended solids and solutes of high molecular weight are retained, while water and low molecular weight solutes pass through the membrane
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32. Ultrafiltration
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33. Conclusion Scale up is the major step of manufacturing
Lab scale technology may not be adapted well with large scale production
Cost, Efficiency and plant layout determined scale up technology
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34. Suggested readings
1. Butler, M. and A. Meneses-Acosta, Recent advances in technology supporting biopharmaceutical production from mammalian cells. Applied Microbiology and Biotechnology, (4): p
2. Warnock, J.N. and M. Al-Rubeai, Bioreactor systems for the production of biopharmaceuticals from animal cells. Biotechnol Appl Biochem, (Pt 1): p
3. Eibl, R., et al., Disposable bioreactors: the current state-of-the-art and recommended applications in biotechnology. Appl Microbiol Biotechnol, (1): p
4. Sukhla, A., et al., Process Scale Bioseparations For The Biopharmaceutical Industry, p
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35. Status of Novel Adsorptive Membranes
Convective mass transfer
No diffusion limitations
Low pressure drop
Best suited for flow-through applications
Impurity removal, polishing
Much higher capacity than porous particles for large targets
Virus, DNA plasmids
Not enough capacity for product capture
Large elution volumes, high dispersion, low product concentration
Pall Mustang
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Sartorius

36. Status of Monolith Technologies
Convective mass transfer – no diffusion limitations
Good dispersion properties, low pressure drops
Larger channels (~2 µm) optimum for large product capture (virus, DNA plasmids, IgM)
IgM capacities reported in the range of mg/mL
Cast as a single unit – Size limitations
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BIA Separations

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39. Thank you for you attention Any Question
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40. Generic Platform Process for Purification of MAbs
Upstream
Downstream
Mammalian Cell Culture Reactor
Cell Removal
Centrifugation or microfiltration
Product Capture
Protein A affinity chromatography
HCP DNA
Cells
Low pH virus inactivation
Purification
CEX or HIC chromatography
Purification
Anion exchange chromatography
Virus Removal
Nanofiltration
Formulation
UF/DF
Aggregates
Degradation products
HCP Protein A
DNA
Yields 60-75%
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41. Figure 1: Errors and heterogeneity in the biotechnological production of protein drugs. Ectopic expression of protein drugs in recombinant host cells, such as bacterial, yeast and mammalian cells may lead to multiple product variants (T, D, A, F, MO, M) due to the operation of multiple physiological and stress response pathways under normal and sub-optimal growth conditions, respectively. Critical parameters for the quality of the resulting product variants may be differentially affected and become further impaired during fermentation in the large scale. The accompanying sub-optimal process parameters will lead to cellular stress due to local nutrient and oxygen depletion or pH and temperature increase. These deviations from the optimal process will not be recognized by sensors operating outside of the bioreactor. Furthermore, bioreactors are susceptible for infiltration by contaminants. A, aggregated protein; D, degraded protein; F, protein with false amino acids incorporated; M, misfolded denatured protein; MO, posttranslationally modified protein; T, prematurely terminated protein.
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