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Platform Technologies to Accelerate Novel Vaccine Development and Manufacturing

Merck KGaA, operating as MilliporeSigma in the US and Canada, focuses on advancing vaccine development and manufacturing through platform technologies, aiming to enhance scalability and speed in response to pandemics. Key insights include the need for a paradigm shift in vaccine processes and the effectiveness of various vaccine modalities, including viral vector and virus-like particle vaccines. Challenges in vaccine production highlight the importance of optimizing bioprocessing and regulatory pathways to ensure rapid and efficient vaccine deployment.

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The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada. Platform Technologies to Accelerate Novel Vaccine Development and Manufacturing David Loong PhD Senior Consultant, Novel Modalities, Asia Pacific, BioProcessing Strategy Josephine Cheng Senior Consultant, Core Modalities, Asia Pacific, BioProcessing Strategy
The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada
Content 1 2 3 Vaccine Platform Technology & Benefits Key platform introduction-Case studies Comparison & summary
Vaccine Platform Technology & Benefits
Eradication of diseases such as smallpox 3 million deaths prevented every year Reduction from 125 to 2 Polio endemic countries in 30 years 5 A preparation that is administered to stimulate the body's immune response against a specific infectious agent or disease Introduction Vaccines 5
Our mission Enabling the industry to produce better faster safer vaccines to improve access, globally Vaccines are a major contribution to overall Global Health 6
Scalability Need to be able to produce huge amount of doses in a short time Global vaccine manufacturing capacity may not be sufficient for COVID19 Time The typical vaccine paradigm doesn’t allow adequate response to tackle outbreaks Cost Vaccine development & licensure requires >$500 million No guaranteed long-term market Major Challenges in Vaccine manufacturing with Outbreak & Pandemic A Paradigm Shift with Pandemics 7
A Paradigm Shift in Vaccine Manufacturing Time is compressed, phases overlap Phase 1 Target ID, development partner selection & Pre-clinical trials Phase 2a Phase 3 Licensure Small-scale clinical trial material Manufacturing scale-up, commercial scale, process validation Large-scale manufacturing Clinical development Target ID, development partner selection & Pre-clinical trials Safety/ Dose selection Safety/efficacy Manufacturing development, scale up, clinical lots, commercial scale, validation of process Large-scale manufacturing Go or no-go decision to invest in candidate First trial in humans Efficacy trial in humans Evaluation trial in humans Go or no-go decision to invest in candidate First in humans (safety) Regulatory pathway for emergency authorization Efficacy trial Traditional paradigm +10years Outbreak paradigm 1-3 years Adapted from NEJM, Lurie et al, March 30th, 2020 8
How they have influenced vaccine manufacturing Pandemics and outbreaks 2021 9
• Global capacity is constrained to respond to COVID19 need • The “unknown” preparedness remain the biggest challenge • Next time, how can we minimize risks and be better prepared?” A Paradigm Shift Lessons learned and Approaches ▪ Acceleration of development is needed. ▪ Prepare for scalability once vaccine is developed. ▪ Platform Technology to reduce changes & increase production capabilities. 10
Inactivated virus Vaccines compose of dead virus DNA vaccines Vaccines compose of snippets of pathogen genes Vaccine by modalities Traditional vaccine Recombinant protein Vaccines with protein or a protein fragment of the pathogen, assembled to closely resemble viruses Conjugated vaccine Vaccines compose of sugars that mimic bacteria pathogen on a carrier protein that increase immune response Viral vector vaccine Vaccines that use a virus to deliver snippets of pathogen genes into human cells Virus-like particles Vaccines with protein or a protein fragment of the pathogen, with a structure closely resemble the pathogen Modern to cutting edge vaccine Live attenuated virus Vaccines compose of weakened virus Toxoid Vaccines compose of inactivated toxin Vaccines compose of inactivated bacteria Whole bacteria RNA vaccines Vaccines compose of snippets of pathogen genes 1921 1924 1945 1955 1986 - 2006 1986 - 1991 1987 - 1991 2019 2020 2021 « One bug – one drug » « One platform – multiple vaccines» Improvements towards effective, safe, and affordable vaccine A vaccine platform is any underlying technology, a mechanism, delivery method, or cell line that can be used to develop multiple vaccines. Replication incompetent Replication competent Various expression systems Synthetic mRNA Self-amplifying (saRNA) 11
Benefits Supports bioprocessing standardization 1 Reduce process development time 2 3 4 Reduce significant manufacturing changes Ease of regulatory approval 12
Key platform introduction: Case studies - VLP, Viral Vector, pDNA, mRNA
Protein Subunit vaccines/ VLP vaccines 14
A VLP has the shape of virus but no genetic materials, good immune response & no risk of pathogenicity Virus Like Particles – a versatile platform • Virus-like particles (VLPs) are biological nanoparticles composed of viral structural proteins, frequently major proteins in the capsid or envelop. • Contain repetitive high-density displays of viral surface proteins that elicit strong immune responses. • Self-assemble into structures morphologically resembling viruses. • No genetic material – no replications, non-infectious. • 20 to 200 nm in size and is similar to the size of the corresponding viruses, allows them to be taken up by dendritic cells (DCs) and antigen-presenting cells (APCs). • Can be produced in a variety of cell culture systems, against different strains of a virus other than those for which the vaccine was formulated. • They sometimes require adjuvants to increase their immunogenicity • Proven technology: Hepatitis B, Human Papillomavirus vaccines 15
VLPs are either non-enveloped or enveloped VLPs can be divided into two groups: Non-enveloped (“naked”) and Enveloped. Each groups could be classified based on the number of viral surface proteins. Non- enveloped HBV, HPV Enveloped CMV Source:Appl Microbiol Biotechnol (2015) 99:10415–10432 Transfus Med Hemother. 2010 Dec; 37(6): 365–375 VLP designs examples (a) 1 layer - 1 proteins Hepatitis B (b) 1 layer- 2 proteins SARS Coronavirus VLPs (c) 2 layers-2 proteins Papillomavirus L1 and L2 (d) 2 layers- multiple proteins FMDV-VLPs (e) 3 layers- multiple proteins Bluetongue virus, rotavirus (f) 1 layer-1 protein Influenza virus (g) 1 layer – 2 proteins Hantaviruses (h) 2 layers- 2 proteins Hepatitis C (j) 2 layers – multiple proteins SARS coronavirus VLP eVLP 16
Production & Purification of VLP-Based Vaccine Insect Cell / Baculovirus VLP Production Platform UF/DF Baculovirus Inactivation Purification Chromatography Media and Inoculum Preparation Cell growth in Bioreactor and Virus Inoculation Bioburden Reduction Primary Clarification Sterile Filtration Polishing Chromatography UF/DF Removal 17
Bacteria Yeast Mammalian Cell Plants Insect Cell VLP Type Non- enveloped Non-enveloped Enveloped Non-enveloped Enveloped Non-enveloped Enveloped Non-enveloped Enveloped Secretory Expression - + ++ + ++ Speed +++ +++ ++ ++ ++ Cost + + +++ ++ ++ Scalability +++ +++ ++ ++ ++ VLP Complexity + ++ ++ ++ +++ Yield +++ ++ + ++ ++ Reported Yields 4.38 g/L Polyomavirus VP1 400 mg/L Hepatitis B Surface Antigen 500 mg/L Adeno-associated Virus 3 g/kg Papillomavirus L1 662 mg/L Rotavirus VP2, VP6 and VP7 Different VLP Production Systems Source: Shiyu Dai et.al. Journal of Immunological Sciences (2018). - No successful cases reported; + Low; ++ Medium; +++ High Linda et al., Biotechnology and Bioengineering, Vol. 111, No. 3, March, 2014 18
VLP Technology Challenges 1 pH, solubility, shear, proteolytic 2 Adsorption Shear filtration chromatography resolution 3 Endotoxin removal in bacterial expression systems Difficulties in baculovirus (BV) removal (VLPs with similar sizes and/or surface charge at a given pH) Stability yields Contaminant Removal 19 Overall, depending on the expression platform of VLPs, the production processes should be designed to accommodate the best compromise between throughput, yield and quality needs meeting the desired purity and potency, and the cost.
1. Disease Target: Hepatitis C  170 million people infected, over 350,000 deaths/year  Causes cirrhosis and liver cancer  Current therapies only partially effective, costly and poorly tolerated  No vaccine currently exists 2. VLP produced in Sf9 insect cells co-infected with MLV-GAG and HCV-E1E2 using baculovirus Purification process optimization for VLP-based Vaccine Case sharing collaboration with iBET iBET: Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal Sf9 ANTIGENS E1 and E2 envelope glycoproteins from Hepatitis C virus STRUCTURE Capsid and envelope from retrovirus (murine leukemia virus) 20
21 Production & Purification of VLP-Based Vaccine Process Challenges Scalable production in bioreactor Efficient purification High recovery of VLP Baculovirus clearance VLP Baculovirus 1 2 3 4
Production & Purification of VLP-Based Vaccine Cell culture Optimization Cell growth obtained using Mobius® 3L Bioreactor Western blot analysis of VLPs using three markers • Comparable cell and VLP properties between disposable and glass bioreactors. • Increased agitation rate, increased cell density of inoculation, replacing the micro- sparger with an open-pipe sparger improved the performance of the single-use bioreactor. • Reproducible performance of the disposable bioreactor. 22
Unlike centrifugation (CFG), depth filtration resulted in ~70% DNA clearance 0% 20% 40% 60% 80% 100% 10 μm → 5 μm → 0.6 μm 10 μm → 0.6 μm 5 μm → 0.6 μm 5 μm → 0.3 μm CFG → 0.6 μm CFG → 0.3 μm CFG VLP recovery HepC VLP clarification Production & Purification of VLP-Based Vaccine Clarification Optimization Clarification • Filter-only clarification train can be used without compromising recovery yield of VLPs. • Filter cascade composed of a Polygard® CN 5 μm filter followed by a 0.3 μm depth filter showed the highest recovery of HCV-VLP, improving on centrifugation/2° depth filtration • Moderate DNA removal with depth filtration was seen 23
24 Virus-Like Paricle Vaccine Training 90% 35% 85% 80% 28% 91% 58% 90% 96% 38% 0% 25% 50% 75% 100% HCV-VLP recovery % BV removal % Total Protein removal % DNA removal % HCP removal% Pellicon® (PES 100 kD) Pellicon® (CRC 300 kD) Both membranes were fully retentive of the VLP Better removal of baculovirus, DNA, and host-cell protein! Optimization of TFF for Concentration and Purification Purification Results UF/DF Pellicon® cassette with 300 kD regenerated cellulose membrane offered the best combination of recovery and purification 4-5X concentration achieved 24
▪ Successfully purified VLPs using Fractogel® TMAE commercial resins ▪ Yield of >60% with ~2 LRV baculovirus can be achieved with a flow-through/wash purification strategy ▪ Options to increase recovery or purification depending on product value by varying process conditions Separation of VLP from Baculovirus Anion Exchange Chromatography for VLP Purification Higher flow rate OR Higher load conductivity Recovery LRV Flow rate (mL/min) NaCl (mM) Higher recovery AND Lower BV LRV Inputs: [NaCl] (100/200/300 mM) and flow rate (100/200/400 cm/hr) Responses: % VLP recovery and Baculovirus LRV Purification Recovery 25
All components were integrated in a templatable and scalable process that made it possible to achieve the desired yield and recovery Summary iBET collaboration • A Mobius® 3L single-use bioreactor was successfully used to produce a VLP-based vaccine in an insect cell culture system • Downstream processing was optimized using Polygard® CN 5.0→0.3 μm depth filters, followed by UF/DF using a Pellicon® cassette with Ultracel® 300 kDa Membrane • VLPs were purified using Fractogel® EMD TMAE resins and Eshmuno® resin prototypes 26
Viral Vector based vaccines 27
Viral Vectors shuffle genetic messages into cells for making target antigens without getting human sick A Trojan-horse Vaccine 1. A live vector vaccine is a a vaccine that uses a weakened or harmless microorganism to transport pieces of antigen in order to stimulate an immune response. 2. Common viral vectors are adenovirus, canarypox, lentivirus, and alphaviruses. 3. They transfect their own DNA into the host cell, which is later expressed to produced new viral particles. 4. Viral vectors are genetically modified to have non- replicating viral vectors (e.g. adenovirus) and replicating viral vectors (e.g. weakened Measles). 5. Benefits including strong immue response, possible to encode several antigens (multi-diseases), relatively inexpensive and stable to transport. Widely used for vaccines and cancer drug. 6. Risk of reversion of virulence, potential cancerous cell cancerous phenotype. 28
Adenovirus based vaccine pipelines Adenovirus (AV) is most commonly used in vaccine development Vaccine Target Type Company Approval year Comments Ad5- EBOV Ebola Ad5 CanSino & Beijing Institute of Biotechnology, China 2017 Lyophilized 1 dose Zabdeno ® Ad26.ZE BOV Ebola AdVac® (Ad26) Johnson & Johnson (Janssen Vaccines) 2020 Liquid frozen 2 doses, boost: Mvabea® (MVA- BN-Filo) with a different vector Vaccine Type Phase Company AZD1222 Adenovirus EUA AstraZeneca & University of Oxford Ad5-nCoV Adenovirus EUA CanSino & China’s military research unit Ad26COVS1 Adenovirus EUA Johnson & Johnson Current approved adenovirus based vaccines Current COVID-19 adenovirus* *As of date July 2021 • Human adenoviruses and many animal adenoviruses (monkeys, cattle, sheep, swine, dogs), belong to the genus Mastadenovirus • Adenovirus in its natural form is a pathogen for common cold. The AV used for vaccine developments are specially genetically modified NOT to cause any diseases and generally regarded as safe. • Non-Enveloped viruses with a ds-DNA, 70-90 nm in size, small enough to go through sterile filtration. 29
Schematic Adenovirus manufacturing process Viral Vector Manufacturing Raw materials Media prep Amplification & inoculation Cell culture Clarification (I & II) Bioburden removal Nucleic acid digestion Concentration Bioburden removal AEX Chrom Concentration & Diafiltration Formulation Sterile filtration Final Filling Lysis SEC or AEX chrom Upstream Downstream Formulation & Fill Finish Downstream 30
Viral Vector Technology Challenges 1 Reaching sufficient viral titers 2 3 Needs to meet regulatory requirements Recovery Cost & scalability 31 Titer Ensure minimum losses in downstream 4Purity & Quality
Simian Adenovirus platform for vaccines 32 Objectives: • GMP process • Easy to operate • Single use • >50% efficiency • Phase 1 scale: >5x1013 VP (1000 doses) • Readily scalable to 5x1014 • Vaccine candidate: Rabies vaccine « ChAdOx2-RabGP »
Transition to a new manufacturing process Reduced handling, compressed process Benzonase® Nuclease Clarification Millistak+® filters Concentration/ Diafiltration Pellicon® 2 cassettes Membrane chromatography Natrix® membrane Diafiltration/ Formulation Pellicon® 2 cassettes Final filtration Durapore® filters Mobius® Bioreactor Centrifugation Ultra-centrifugation Shake flasks 33
Conclusion Accelerating vaccine development & manufacturing is possible GMP ready for various adenoviruses Full single use process Project met initial goals >2000 doses/4L culture 18 months project Collaboration between supplier/ manufacturer is key 5 days process time: « outbreak » ready 34
What is the advantage of a platform? The Need to Platform 1. Supports bioprocessing standardization 2. Reduce process development time 3. Reduce significant manufacturing changes 4. Ease regulatory approval At the Jenner Institute, in 2 months, process development was done based on the previously developed platform Optimization of critical unit operations were done hand in hand with Merck engineers 35
Nucleic Acid Vaccine – pDNA & mRNA 36
The rise of nucleic acid technologies Nucleic acids require a delivery system to be effective Naked Encapsulated DNA RNA Target gene DNA Plasmid DNA is the common approach mRNA Lipid nanoparticles formation using lipids and/or polymers to protect from nucleases & endosomes Intravenous or lymphatic injection route siRNA Antisense RNA Local administration at target site Rapid degradation Intratumoral, intranodal or subq Other types of RNA 37
mRNA technology Two types of mRNA constructs are being actively used Non-Replicating mRNA (NRM) Self Amplifying mRNA (SAM) Number of nucleotides 2000 – 3000 nt ~ 10 000 nt Type Single-stranded Single-stranded Size 660 – 990 kDa 3300 kDa Potency Low levels of proteins High levels of proteins Enhanced protein expression Immunity No theoretical risk of anti-vector immunity with non-viral delivery systems No anti-vector effect has been observed yet Potential interactions between encoded non-structural proteins and host factors require additional investigation. Concentration needed/dose ~50-200ug/dose ~1ug/dose 5’ UTR GOI 3’ UTR 5’ cap A A A A A A A A 5’ UTR GOI 3’ UTR 5’ cap A A A A A A A A Replicase 38
mRNA technology Advantages 1 RNA therapeutics are safer than DNA therapeutics (RNA does not integrate into the Genom) RNA is not infectious RNA is produced using a cell-free enzymatic transcription reaction or chemical synthesis 2 Production of RNA- based vaccines is faster compared to production of traditional vaccines Good scalability 3 Producing RNA vaccines is less expensive than producing the full antigen protein 4 For any outbreak RNA vaccines are more flexibel, any desired RNA for any desired protein of interest can be prepared in short time for each individual patient (personalized medicine) Safety Time COst Flexibility 39 39
mRNA technology Challenges 1 Single stranded Highly negatively charged Rapid degradation of RNA caused by endonucleases Cold-chain 2 Exogenous mRNA is immunostimulatory, as it is recognized by a variety of innate immune receptors In applications such as protein-replacement therapies, activation of the innate immune system by toll-like receptors by IVT mRNA is not desired 3 The in vivo Delivery of RNA is very inefficient RNA vaccines have a lower immunogenicity compared to traditional vaccines, so higher doses are needed RNA instability Immune modulation Efficiency 40 40
Generate pDNA coding for the RNA polymerase promoter and the targeted mRNA construct mRNA manufacturing starts with a plasmid template Plasmid process Fermentation Clarification Purification with Chrom (2-3 steps) Final Filtration Concentration and Diafiltration (UF/DF) Thaw cells E.coli+pDNA Cell Harvest Cell Lysis Concentration and Diafiltration (UF/DF) Plasmid facility mRNA facility pDNA Non replicating mRNA Self amplifying mRNA pDNA 41
High-performing and single-use chromatography devices simplify the process Plasmid process • Superior productivity and flexibility for multi- product/multi-purpose manufacturing of plasmids • Ideally suited for the capture of small and mid-sized Plasmids (<20kbp) • Yield: ≥80 % of ccc-form, >95 % RNA removal • Short cycle time: 35 min • Caustic stability enables efficient cleaning (1M NaOH) • Re-use in rapid-cycling operation mode allows for cost reduction Natrix® Q Chromatography membrane Plasmid Size Plasmid Titer µg/ml Initial pDNA Purity A260 based Operating Capacity mg/mL MV Yield % pDNA Eluate Purity A260 based 5.7 kb 45 4.0 %  10 ≥ 80 ≥ 80 % pDNA 13 kb 33 2.4 %  4 ≥ 77 ≥ 90 % pDNA 20 kb 25 0.7 % 1 ≥ 65 ≥ 62 % pDNA Case study Feed characteristics • Original E. coli lysates from alkaline lysis, clarified by centrifugation/depth filtration, supplemented with optimal NaCl to eliminate RNA interference, pH 5.0, finally 0.22 µm filtered • Plasmids ranging from small to large size • Varying initial pDNA purity ranging from 0.7% - 4% Results 42
mRNA manufacturing Scale-up considerations Lab scale • Solvent extraction • Precipitation steps • Hazardous solvents • Scalability issues • Lack of process development expertise • Replace extraction & precipitation with chrom, TFF • GMP compliance • Risk assessment for Rnase-free biopharma materials, process equipment, raw material & solutions • Complex development of efficient & safe encapsulation systems • Challenges in sterile filtration of large mRNA complexes • Cold-chain distribution to point of care Manufacturing Scale Make Purify Formulate pDNA Linearization Chrom, UF/DF In vitro Transcription Chrom, UF/DF Enzymatic capping Chromatography UF/DF Encapsulation & Formulation Final Sterile Filtration 43
mRNA manufacturing Process objectives: quality Make Purify Formulate pDNA Linearization Chrom, UF/DF In vitro Transcription Chrom, UF/DF Enzymatic capping Chromatography UF/DF Encapsulation & Formulation Final Sterile Filtration mRNA product • mRNA Size/MW • mRNA integrity, potency • Encapsulation efficiency • Capping efficiency • Impurities: dsRNA, DNA template, nucleoside triphosphates, RNA polymerase • Appearance, pH, osmolality, subvisible particles, elemental impurities and residual solvents Quality Control Quality Attributes mRNA structure Purity Low impurities promotes mRNA expression • 5’ Cap: Affect innate sensing and protein • UTRs: Maximize gene expression • GOI or Coding Sequence Region Gene of Interest • 3’ Poly-A-tail: Enhances translation & protects mRNA Efficient delivery system (nanoparticle) 44
mRNA formulation Encapsulation is crucial for mRNA stability and delivery efficiency Drug Delivery Technologies Lipids Lipid Nanoparticle (LNP) Polymers Liposomes Lipoplexes Polyplexes mRNA in aqueous solution Sterile Filtration & Fill and Finish mRNA LNP formation LNP Formulation UF/DF & Final formulation Encapsulation Lipids in organic solvent mRNA LNP rug product LNP is most commonly used for mRNA delivery Each LNP consists of four different lipids allowing the mRNA to be carried in it and protected from degradation 45
Comparison & summary
COVID pipeline analysis (July.15,2021) 1 1 2 2 19 18 9 16 21 24 71 1 2 2 2 5 16 10 16 18 36 0 20 40 60 80 100 120 VVnr + Antigen Presenting Cell Cellular based vaccine Bacterial vector (Replicating) VVr + Antigen Presenting Cell Live attenuated bacterial vector Live attenuated virus Viral vector (Replicating) Virus like particle Inactivated virus DNA based vaccine Viral vector (Non-replicating) RNA based vaccine Protein subunit Total Pipelines in COVID-19 Vaccine Pre-Clinical Clinical Trials PS > VV > mRNA > pDNA > IV > VLP 47
Summary Platform technology can reduce the development time and cost, whilst minimizing process changes between vaccines. Platform technologies can provide high level of flexibility and can be quickly adjusted for variants especially in pandemic and outbreaks. Choosing which platform to use depends on available assets and resources, overall cost, targeted diseases, etc. All products going forward needs to approve safety and efficacy by the regulatory authorities. 1 2 3 48 4
Fedosiuk et al. Silva et al. Peixoto et al. Dr Anissa Boumlic Elina Gousseinov Claire Scanlan Thomas Parker Nargisse El Hajjami Manuel Brantner Matthieu Perret Sandra Hon Acknowlegement: Thank you for listening! 49
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Platform Technologies to Accelerate Novel Vaccine Development and Manufacturing

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Platform Technologies to Accelerate Novel Vaccine Development and Manufacturing

  • 1.
    The life sciencebusiness of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada. Platform Technologies to Accelerate Novel Vaccine Development and Manufacturing David Loong PhD Senior Consultant, Novel Modalities, Asia Pacific, BioProcessing Strategy Josephine Cheng Senior Consultant, Core Modalities, Asia Pacific, BioProcessing Strategy
  • 2.
    The life sciencebusiness of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada
  • 3.
    Content 1 2 3 Vaccine Platform Technology& Benefits Key platform introduction-Case studies Comparison & summary
  • 4.
  • 5.
    Eradication of diseases suchas smallpox 3 million deaths prevented every year Reduction from 125 to 2 Polio endemic countries in 30 years 5 A preparation that is administered to stimulate the body's immune response against a specific infectious agent or disease Introduction Vaccines 5
  • 6.
    Our mission Enabling theindustry to produce better faster safer vaccines to improve access, globally Vaccines are a major contribution to overall Global Health 6
  • 7.
    Scalability Need to beable to produce huge amount of doses in a short time Global vaccine manufacturing capacity may not be sufficient for COVID19 Time The typical vaccine paradigm doesn’t allow adequate response to tackle outbreaks Cost Vaccine development & licensure requires >$500 million No guaranteed long-term market Major Challenges in Vaccine manufacturing with Outbreak & Pandemic A Paradigm Shift with Pandemics 7
  • 8.
    A Paradigm Shiftin Vaccine Manufacturing Time is compressed, phases overlap Phase 1 Target ID, development partner selection & Pre-clinical trials Phase 2a Phase 3 Licensure Small-scale clinical trial material Manufacturing scale-up, commercial scale, process validation Large-scale manufacturing Clinical development Target ID, development partner selection & Pre-clinical trials Safety/ Dose selection Safety/efficacy Manufacturing development, scale up, clinical lots, commercial scale, validation of process Large-scale manufacturing Go or no-go decision to invest in candidate First trial in humans Efficacy trial in humans Evaluation trial in humans Go or no-go decision to invest in candidate First in humans (safety) Regulatory pathway for emergency authorization Efficacy trial Traditional paradigm +10years Outbreak paradigm 1-3 years Adapted from NEJM, Lurie et al, March 30th, 2020 8
  • 9.
    How they haveinfluenced vaccine manufacturing Pandemics and outbreaks 2021 9
  • 10.
    • Global capacityis constrained to respond to COVID19 need • The “unknown” preparedness remain the biggest challenge • Next time, how can we minimize risks and be better prepared?” A Paradigm Shift Lessons learned and Approaches ▪ Acceleration of development is needed. ▪ Prepare for scalability once vaccine is developed. ▪ Platform Technology to reduce changes & increase production capabilities. 10
  • 11.
    Inactivated virus Vaccines compose of deadvirus DNA vaccines Vaccines compose of snippets of pathogen genes Vaccine by modalities Traditional vaccine Recombinant protein Vaccines with protein or a protein fragment of the pathogen, assembled to closely resemble viruses Conjugated vaccine Vaccines compose of sugars that mimic bacteria pathogen on a carrier protein that increase immune response Viral vector vaccine Vaccines that use a virus to deliver snippets of pathogen genes into human cells Virus-like particles Vaccines with protein or a protein fragment of the pathogen, with a structure closely resemble the pathogen Modern to cutting edge vaccine Live attenuated virus Vaccines compose of weakened virus Toxoid Vaccines compose of inactivated toxin Vaccines compose of inactivated bacteria Whole bacteria RNA vaccines Vaccines compose of snippets of pathogen genes 1921 1924 1945 1955 1986 - 2006 1986 - 1991 1987 - 1991 2019 2020 2021 « One bug – one drug » « One platform – multiple vaccines» Improvements towards effective, safe, and affordable vaccine A vaccine platform is any underlying technology, a mechanism, delivery method, or cell line that can be used to develop multiple vaccines. Replication incompetent Replication competent Various expression systems Synthetic mRNA Self-amplifying (saRNA) 11
  • 12.
  • 13.
    Key platform introduction: Casestudies - VLP, Viral Vector, pDNA, mRNA
  • 14.
  • 15.
    A VLP hasthe shape of virus but no genetic materials, good immune response & no risk of pathogenicity Virus Like Particles – a versatile platform • Virus-like particles (VLPs) are biological nanoparticles composed of viral structural proteins, frequently major proteins in the capsid or envelop. • Contain repetitive high-density displays of viral surface proteins that elicit strong immune responses. • Self-assemble into structures morphologically resembling viruses. • No genetic material – no replications, non-infectious. • 20 to 200 nm in size and is similar to the size of the corresponding viruses, allows them to be taken up by dendritic cells (DCs) and antigen-presenting cells (APCs). • Can be produced in a variety of cell culture systems, against different strains of a virus other than those for which the vaccine was formulated. • They sometimes require adjuvants to increase their immunogenicity • Proven technology: Hepatitis B, Human Papillomavirus vaccines 15
  • 16.
    VLPs are eithernon-enveloped or enveloped VLPs can be divided into two groups: Non-enveloped (“naked”) and Enveloped. Each groups could be classified based on the number of viral surface proteins. Non- enveloped HBV, HPV Enveloped CMV Source:Appl Microbiol Biotechnol (2015) 99:10415–10432 Transfus Med Hemother. 2010 Dec; 37(6): 365–375 VLP designs examples (a) 1 layer - 1 proteins Hepatitis B (b) 1 layer- 2 proteins SARS Coronavirus VLPs (c) 2 layers-2 proteins Papillomavirus L1 and L2 (d) 2 layers- multiple proteins FMDV-VLPs (e) 3 layers- multiple proteins Bluetongue virus, rotavirus (f) 1 layer-1 protein Influenza virus (g) 1 layer – 2 proteins Hantaviruses (h) 2 layers- 2 proteins Hepatitis C (j) 2 layers – multiple proteins SARS coronavirus VLP eVLP 16
  • 17.
    Production & Purificationof VLP-Based Vaccine Insect Cell / Baculovirus VLP Production Platform UF/DF Baculovirus Inactivation Purification Chromatography Media and Inoculum Preparation Cell growth in Bioreactor and Virus Inoculation Bioburden Reduction Primary Clarification Sterile Filtration Polishing Chromatography UF/DF Removal 17
  • 18.
    Bacteria Yeast Mammalian Cell PlantsInsect Cell VLP Type Non- enveloped Non-enveloped Enveloped Non-enveloped Enveloped Non-enveloped Enveloped Non-enveloped Enveloped Secretory Expression - + ++ + ++ Speed +++ +++ ++ ++ ++ Cost + + +++ ++ ++ Scalability +++ +++ ++ ++ ++ VLP Complexity + ++ ++ ++ +++ Yield +++ ++ + ++ ++ Reported Yields 4.38 g/L Polyomavirus VP1 400 mg/L Hepatitis B Surface Antigen 500 mg/L Adeno-associated Virus 3 g/kg Papillomavirus L1 662 mg/L Rotavirus VP2, VP6 and VP7 Different VLP Production Systems Source: Shiyu Dai et.al. Journal of Immunological Sciences (2018). - No successful cases reported; + Low; ++ Medium; +++ High Linda et al., Biotechnology and Bioengineering, Vol. 111, No. 3, March, 2014 18
  • 19.
    VLP Technology Challenges 1 pH,solubility, shear, proteolytic 2 Adsorption Shear filtration chromatography resolution 3 Endotoxin removal in bacterial expression systems Difficulties in baculovirus (BV) removal (VLPs with similar sizes and/or surface charge at a given pH) Stability yields Contaminant Removal 19 Overall, depending on the expression platform of VLPs, the production processes should be designed to accommodate the best compromise between throughput, yield and quality needs meeting the desired purity and potency, and the cost.
  • 20.
    1. Disease Target:Hepatitis C  170 million people infected, over 350,000 deaths/year  Causes cirrhosis and liver cancer  Current therapies only partially effective, costly and poorly tolerated  No vaccine currently exists 2. VLP produced in Sf9 insect cells co-infected with MLV-GAG and HCV-E1E2 using baculovirus Purification process optimization for VLP-based Vaccine Case sharing collaboration with iBET iBET: Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal Sf9 ANTIGENS E1 and E2 envelope glycoproteins from Hepatitis C virus STRUCTURE Capsid and envelope from retrovirus (murine leukemia virus) 20
  • 21.
    21 Production & Purificationof VLP-Based Vaccine Process Challenges Scalable production in bioreactor Efficient purification High recovery of VLP Baculovirus clearance VLP Baculovirus 1 2 3 4
  • 22.
    Production & Purificationof VLP-Based Vaccine Cell culture Optimization Cell growth obtained using Mobius® 3L Bioreactor Western blot analysis of VLPs using three markers • Comparable cell and VLP properties between disposable and glass bioreactors. • Increased agitation rate, increased cell density of inoculation, replacing the micro- sparger with an open-pipe sparger improved the performance of the single-use bioreactor. • Reproducible performance of the disposable bioreactor. 22
  • 23.
    Unlike centrifugation (CFG), depthfiltration resulted in ~70% DNA clearance 0% 20% 40% 60% 80% 100% 10 μm → 5 μm → 0.6 μm 10 μm → 0.6 μm 5 μm → 0.6 μm 5 μm → 0.3 μm CFG → 0.6 μm CFG → 0.3 μm CFG VLP recovery HepC VLP clarification Production & Purification of VLP-Based Vaccine Clarification Optimization Clarification • Filter-only clarification train can be used without compromising recovery yield of VLPs. • Filter cascade composed of a Polygard® CN 5 μm filter followed by a 0.3 μm depth filter showed the highest recovery of HCV-VLP, improving on centrifugation/2° depth filtration • Moderate DNA removal with depth filtration was seen 23
  • 24.
    24 Virus-Like Paricle VaccineTraining 90% 35% 85% 80% 28% 91% 58% 90% 96% 38% 0% 25% 50% 75% 100% HCV-VLP recovery % BV removal % Total Protein removal % DNA removal % HCP removal% Pellicon® (PES 100 kD) Pellicon® (CRC 300 kD) Both membranes were fully retentive of the VLP Better removal of baculovirus, DNA, and host-cell protein! Optimization of TFF for Concentration and Purification Purification Results UF/DF Pellicon® cassette with 300 kD regenerated cellulose membrane offered the best combination of recovery and purification 4-5X concentration achieved 24
  • 25.
    ▪ Successfully purifiedVLPs using Fractogel® TMAE commercial resins ▪ Yield of >60% with ~2 LRV baculovirus can be achieved with a flow-through/wash purification strategy ▪ Options to increase recovery or purification depending on product value by varying process conditions Separation of VLP from Baculovirus Anion Exchange Chromatography for VLP Purification Higher flow rate OR Higher load conductivity Recovery LRV Flow rate (mL/min) NaCl (mM) Higher recovery AND Lower BV LRV Inputs: [NaCl] (100/200/300 mM) and flow rate (100/200/400 cm/hr) Responses: % VLP recovery and Baculovirus LRV Purification Recovery 25
  • 26.
    All components were integratedin a templatable and scalable process that made it possible to achieve the desired yield and recovery Summary iBET collaboration • A Mobius® 3L single-use bioreactor was successfully used to produce a VLP-based vaccine in an insect cell culture system • Downstream processing was optimized using Polygard® CN 5.0→0.3 μm depth filters, followed by UF/DF using a Pellicon® cassette with Ultracel® 300 kDa Membrane • VLPs were purified using Fractogel® EMD TMAE resins and Eshmuno® resin prototypes 26
  • 27.
  • 28.
    Viral Vectors shufflegenetic messages into cells for making target antigens without getting human sick A Trojan-horse Vaccine 1. A live vector vaccine is a a vaccine that uses a weakened or harmless microorganism to transport pieces of antigen in order to stimulate an immune response. 2. Common viral vectors are adenovirus, canarypox, lentivirus, and alphaviruses. 3. They transfect their own DNA into the host cell, which is later expressed to produced new viral particles. 4. Viral vectors are genetically modified to have non- replicating viral vectors (e.g. adenovirus) and replicating viral vectors (e.g. weakened Measles). 5. Benefits including strong immue response, possible to encode several antigens (multi-diseases), relatively inexpensive and stable to transport. Widely used for vaccines and cancer drug. 6. Risk of reversion of virulence, potential cancerous cell cancerous phenotype. 28
  • 29.
    Adenovirus based vaccinepipelines Adenovirus (AV) is most commonly used in vaccine development Vaccine Target Type Company Approval year Comments Ad5- EBOV Ebola Ad5 CanSino & Beijing Institute of Biotechnology, China 2017 Lyophilized 1 dose Zabdeno ® Ad26.ZE BOV Ebola AdVac® (Ad26) Johnson & Johnson (Janssen Vaccines) 2020 Liquid frozen 2 doses, boost: Mvabea® (MVA- BN-Filo) with a different vector Vaccine Type Phase Company AZD1222 Adenovirus EUA AstraZeneca & University of Oxford Ad5-nCoV Adenovirus EUA CanSino & China’s military research unit Ad26COVS1 Adenovirus EUA Johnson & Johnson Current approved adenovirus based vaccines Current COVID-19 adenovirus* *As of date July 2021 • Human adenoviruses and many animal adenoviruses (monkeys, cattle, sheep, swine, dogs), belong to the genus Mastadenovirus • Adenovirus in its natural form is a pathogen for common cold. The AV used for vaccine developments are specially genetically modified NOT to cause any diseases and generally regarded as safe. • Non-Enveloped viruses with a ds-DNA, 70-90 nm in size, small enough to go through sterile filtration. 29
  • 30.
    Schematic Adenovirus manufacturingprocess Viral Vector Manufacturing Raw materials Media prep Amplification & inoculation Cell culture Clarification (I & II) Bioburden removal Nucleic acid digestion Concentration Bioburden removal AEX Chrom Concentration & Diafiltration Formulation Sterile filtration Final Filling Lysis SEC or AEX chrom Upstream Downstream Formulation & Fill Finish Downstream 30
  • 31.
    Viral Vector Technology Challenges 1Reaching sufficient viral titers 2 3 Needs to meet regulatory requirements Recovery Cost & scalability 31 Titer Ensure minimum losses in downstream 4Purity & Quality
  • 32.
    Simian Adenovirus platform forvaccines 32 Objectives: • GMP process • Easy to operate • Single use • >50% efficiency • Phase 1 scale: >5x1013 VP (1000 doses) • Readily scalable to 5x1014 • Vaccine candidate: Rabies vaccine « ChAdOx2-RabGP »
  • 33.
    Transition to anew manufacturing process Reduced handling, compressed process Benzonase® Nuclease Clarification Millistak+® filters Concentration/ Diafiltration Pellicon® 2 cassettes Membrane chromatography Natrix® membrane Diafiltration/ Formulation Pellicon® 2 cassettes Final filtration Durapore® filters Mobius® Bioreactor Centrifugation Ultra-centrifugation Shake flasks 33
  • 34.
    Conclusion Accelerating vaccine development& manufacturing is possible GMP ready for various adenoviruses Full single use process Project met initial goals >2000 doses/4L culture 18 months project Collaboration between supplier/ manufacturer is key 5 days process time: « outbreak » ready 34
  • 35.
    What is theadvantage of a platform? The Need to Platform 1. Supports bioprocessing standardization 2. Reduce process development time 3. Reduce significant manufacturing changes 4. Ease regulatory approval At the Jenner Institute, in 2 months, process development was done based on the previously developed platform Optimization of critical unit operations were done hand in hand with Merck engineers 35
  • 36.
  • 37.
    The rise ofnucleic acid technologies Nucleic acids require a delivery system to be effective Naked Encapsulated DNA RNA Target gene DNA Plasmid DNA is the common approach mRNA Lipid nanoparticles formation using lipids and/or polymers to protect from nucleases & endosomes Intravenous or lymphatic injection route siRNA Antisense RNA Local administration at target site Rapid degradation Intratumoral, intranodal or subq Other types of RNA 37
  • 38.
    mRNA technology Two typesof mRNA constructs are being actively used Non-Replicating mRNA (NRM) Self Amplifying mRNA (SAM) Number of nucleotides 2000 – 3000 nt ~ 10 000 nt Type Single-stranded Single-stranded Size 660 – 990 kDa 3300 kDa Potency Low levels of proteins High levels of proteins Enhanced protein expression Immunity No theoretical risk of anti-vector immunity with non-viral delivery systems No anti-vector effect has been observed yet Potential interactions between encoded non-structural proteins and host factors require additional investigation. Concentration needed/dose ~50-200ug/dose ~1ug/dose 5’ UTR GOI 3’ UTR 5’ cap A A A A A A A A 5’ UTR GOI 3’ UTR 5’ cap A A A A A A A A Replicase 38
  • 39.
    mRNA technology Advantages 1 RNAtherapeutics are safer than DNA therapeutics (RNA does not integrate into the Genom) RNA is not infectious RNA is produced using a cell-free enzymatic transcription reaction or chemical synthesis 2 Production of RNA- based vaccines is faster compared to production of traditional vaccines Good scalability 3 Producing RNA vaccines is less expensive than producing the full antigen protein 4 For any outbreak RNA vaccines are more flexibel, any desired RNA for any desired protein of interest can be prepared in short time for each individual patient (personalized medicine) Safety Time COst Flexibility 39 39
  • 40.
    mRNA technology Challenges 1 Singlestranded Highly negatively charged Rapid degradation of RNA caused by endonucleases Cold-chain 2 Exogenous mRNA is immunostimulatory, as it is recognized by a variety of innate immune receptors In applications such as protein-replacement therapies, activation of the innate immune system by toll-like receptors by IVT mRNA is not desired 3 The in vivo Delivery of RNA is very inefficient RNA vaccines have a lower immunogenicity compared to traditional vaccines, so higher doses are needed RNA instability Immune modulation Efficiency 40 40
  • 41.
    Generate pDNA codingfor the RNA polymerase promoter and the targeted mRNA construct mRNA manufacturing starts with a plasmid template Plasmid process Fermentation Clarification Purification with Chrom (2-3 steps) Final Filtration Concentration and Diafiltration (UF/DF) Thaw cells E.coli+pDNA Cell Harvest Cell Lysis Concentration and Diafiltration (UF/DF) Plasmid facility mRNA facility pDNA Non replicating mRNA Self amplifying mRNA pDNA 41
  • 42.
    High-performing and single-usechromatography devices simplify the process Plasmid process • Superior productivity and flexibility for multi- product/multi-purpose manufacturing of plasmids • Ideally suited for the capture of small and mid-sized Plasmids (<20kbp) • Yield: ≥80 % of ccc-form, >95 % RNA removal • Short cycle time: 35 min • Caustic stability enables efficient cleaning (1M NaOH) • Re-use in rapid-cycling operation mode allows for cost reduction Natrix® Q Chromatography membrane Plasmid Size Plasmid Titer µg/ml Initial pDNA Purity A260 based Operating Capacity mg/mL MV Yield % pDNA Eluate Purity A260 based 5.7 kb 45 4.0 %  10 ≥ 80 ≥ 80 % pDNA 13 kb 33 2.4 %  4 ≥ 77 ≥ 90 % pDNA 20 kb 25 0.7 % 1 ≥ 65 ≥ 62 % pDNA Case study Feed characteristics • Original E. coli lysates from alkaline lysis, clarified by centrifugation/depth filtration, supplemented with optimal NaCl to eliminate RNA interference, pH 5.0, finally 0.22 µm filtered • Plasmids ranging from small to large size • Varying initial pDNA purity ranging from 0.7% - 4% Results 42
  • 43.
    mRNA manufacturing Scale-up considerations Labscale • Solvent extraction • Precipitation steps • Hazardous solvents • Scalability issues • Lack of process development expertise • Replace extraction & precipitation with chrom, TFF • GMP compliance • Risk assessment for Rnase-free biopharma materials, process equipment, raw material & solutions • Complex development of efficient & safe encapsulation systems • Challenges in sterile filtration of large mRNA complexes • Cold-chain distribution to point of care Manufacturing Scale Make Purify Formulate pDNA Linearization Chrom, UF/DF In vitro Transcription Chrom, UF/DF Enzymatic capping Chromatography UF/DF Encapsulation & Formulation Final Sterile Filtration 43
  • 44.
    mRNA manufacturing Process objectives:quality Make Purify Formulate pDNA Linearization Chrom, UF/DF In vitro Transcription Chrom, UF/DF Enzymatic capping Chromatography UF/DF Encapsulation & Formulation Final Sterile Filtration mRNA product • mRNA Size/MW • mRNA integrity, potency • Encapsulation efficiency • Capping efficiency • Impurities: dsRNA, DNA template, nucleoside triphosphates, RNA polymerase • Appearance, pH, osmolality, subvisible particles, elemental impurities and residual solvents Quality Control Quality Attributes mRNA structure Purity Low impurities promotes mRNA expression • 5’ Cap: Affect innate sensing and protein • UTRs: Maximize gene expression • GOI or Coding Sequence Region Gene of Interest • 3’ Poly-A-tail: Enhances translation & protects mRNA Efficient delivery system (nanoparticle) 44
  • 45.
    mRNA formulation Encapsulation iscrucial for mRNA stability and delivery efficiency Drug Delivery Technologies Lipids Lipid Nanoparticle (LNP) Polymers Liposomes Lipoplexes Polyplexes mRNA in aqueous solution Sterile Filtration & Fill and Finish mRNA LNP formation LNP Formulation UF/DF & Final formulation Encapsulation Lipids in organic solvent mRNA LNP rug product LNP is most commonly used for mRNA delivery Each LNP consists of four different lipids allowing the mRNA to be carried in it and protected from degradation 45
  • 46.
  • 47.
    COVID pipeline analysis (July.15,2021) 1 1 2 2 19 18 9 16 21 24 71 1 2 2 2 5 16 10 16 18 36 020 40 60 80 100 120 VVnr + Antigen Presenting Cell Cellular based vaccine Bacterial vector (Replicating) VVr + Antigen Presenting Cell Live attenuated bacterial vector Live attenuated virus Viral vector (Replicating) Virus like particle Inactivated virus DNA based vaccine Viral vector (Non-replicating) RNA based vaccine Protein subunit Total Pipelines in COVID-19 Vaccine Pre-Clinical Clinical Trials PS > VV > mRNA > pDNA > IV > VLP 47
  • 48.
    Summary Platform technology canreduce the development time and cost, whilst minimizing process changes between vaccines. Platform technologies can provide high level of flexibility and can be quickly adjusted for variants especially in pandemic and outbreaks. Choosing which platform to use depends on available assets and resources, overall cost, targeted diseases, etc. All products going forward needs to approve safety and efficacy by the regulatory authorities. 1 2 3 48 4
  • 49.
    Fedosiuk et al. Silvaet al. Peixoto et al. Dr Anissa Boumlic Elina Gousseinov Claire Scanlan Thomas Parker Nargisse El Hajjami Manuel Brantner Matthieu Perret Sandra Hon Acknowlegement: Thank you for listening! 49
  • 50.