Manufacturing process of biological products: drug substance : Filtration, Dialysis Inactivation Wisit Tangkeangsirisin, PhD. Biopharmacy Department Faculty of Pharmacy Silpakorn University 11/10/2018

Lecture Outlines Introduction Conclusion Recombinant Proteins significance in Pharmacy Feature of Recombinant Protein Upstream Process technology Filtration/Concentration Inactivation Conclusion 11/10/2018

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).

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.

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

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

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

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

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 0.009 % formaldehyde (i.e., 3.3 mM or 100 μg/ml formaldehyde) 11/10/2018

Inactivation by Formalin in Flavivirus vaccine JE and TBE No specify inactivation condition from WHO or Ph Eur Examples are available (JEV 50-60 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). 11/10/2018

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 11/10/2018

Formaline Inactivation Methods for formaldehyde inactivation vary greatly between vaccines. Differences lie in formalin concentrations (from 0.08 to 0.009 % w/v), time of inactivation (from days to months), and temperature (usually 4 or 37 °C) Now in IIV 11/10/2018

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) 11/10/2018

Overview of FDA approved inactivate influenza vaccines 11/10/2018

Other inactivation methods in development Hydrogen peroxide Zinc finger reactive compounds (in RSV vaccine) Gamma radiation 11/10/2018

Inactivation 11/10/2018

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Filtration 11/10/2018

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) 11/10/2018

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Filtration: Classification Surface Filtration Depth Filtration 11/10/2018

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 11/10/2018

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 11/10/2018

11/10/2018 http://www.induceramic.com/porous-ceramics-application/filtration-separation-application

Cell Isolation/Harvesting Cells Cell Concentrate Membrane Cell Suspension Cell-free culture medium Cell-free culture medium Dead End Filtration Cross Flow Filtration 11/10/2018

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Ultrafiltration/Diafiltration 11/10/2018

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 11/10/2018

Ultrafiltration 11/10/2018

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 11/10/2018

Suggested readings 1. Butler, M. and A. Meneses-Acosta, Recent advances in technology supporting biopharmaceutical production from mammalian cells. Applied Microbiology and Biotechnology, 2012. 96(4): p. 885-894. 2. Warnock, J.N. and M. Al-Rubeai, Bioreactor systems for the production of biopharmaceuticals from animal cells. Biotechnol Appl Biochem, 2006. 45(Pt 1): p. 1-12. 3. Eibl, R., et al., Disposable bioreactors: the current state-of-the-art and recommended applications in biotechnology. Appl Microbiol Biotechnol, 2010. 86(1): p. 41-9. 4. Sukhla, A., et al., Process Scale Bioseparations For The Biopharmaceutical Industry, 2007. p.63-79. 11/10/2018

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 11/10/2018 Sartorius

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 40-50 mg/mL Cast as a single unit – Size limitations 11/10/2018 BIA Separations

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Thank you for you attention Any Question 11/10/2018

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% 11/10/2018

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. http://www.omicsonline.org/2155-9821/images/2155-9821-2-115-g001.gif 11/10/2018

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