Controls and containment in plant-based vaccine manufacturing

1. Controls and containment in plant-based vaccine manufacturing Todd Talarico Vice President of Process Development and Clinical Manufacturing

2. Objective • Introduce Plant BasedBiologics • PresentControlsused for Manufacturingbasedupon: • Health Canada : Guidance Document – Submission Information for Human-Use BiologicalDrugsDerivedfrom Plant MolecularFarming (PMF) • FDA: Draft Guidance for Industry: Drugs, Biologics and MedicalDevicesDerivedfromBioengineered Plants for Use in Humans and Animals • ICH Q7 - Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients • ICH Q5 series – Quality of BiotechnologicalProducts • EMEA – Guideline on the Quality of Biological Active Substances Produced by Stable Transgene Expression in Higher Plants • Guideline on Good Agricultural and Collection Practice (GACP) • Focus on the process first and thencontainment • FamiliarPrinciples • Principlesthatmaybemore unique to plant derivedbiotherapeuticsand vaccines

3. Medicago Overview 3

4. Plant Platforms for the Production of Biologics • Plant cells (bioreactor) • Whole plants • Expression systems • Transgenic plants withnuclear transformation • Transient expression • Induced by plant specificbacteriumor virus polynucleotidetransfer • Advantages of transient expression system • No propagation throughseed stock • Geneticstabilityof expression • Growthsystems • Indoors (Greenhouses, Growth Chambers) • Outdoorswithrestrictions • Expression • Indoors (Greenhouses, Chambers) or Outdoors

5. Medicago’s Technology Plants used for production are not genetically modified and grown in a greenhouse Use a genetically engineered Agrobacterium to drive expression of a protein in the leaves of “tobacco” plants In the case of the Influenza vaccine, the plants produce virus like particles (VLP) containing the HA protein imbedded in a lipid bilayer when incubated under controlled conditions The VLP are extracted from the plant tissue The VLP are purified in a “traditional” bioprocessing scheme to produce a sterile product, suitable for injection Medicago’s Bioreactor 5

6. Key Aspects of Medicago’sProcess Control • Stock maintenance • Plant seed stock maintenance system • Agrobacterium MCB/WCB maintenance • Upstream • Greenhouse • Stock to harvest • Before infiltration • Definedbiologicalstartingmaterial • After infiltration (protein expression) • Downstream • After extraction • Purification, Formulation and Filling • Facility Control • Equipment Control • Personnel Control • RawMaterial Control

7. Raw Material Utilities Personnel Production areas etc The Monovalent VLP DS Process Control Strategy was developed to minimize the variability caused by input parameters in accordance with the principles found in the CMC-Vaccines Working Group document, A-VAX: Applying Quality by Design to Vaccines, issued in May 2012.

8. Guidance Documentsthat support the plant platform • Health Canada : Guidance Document – Plant MolecularFarming (PMF) Applications: Plant-DerivedBiologicalDrugsfor Human Use (2014) • FDA: Draft Guidance for Industry: Drugs, Biologics and MedicalDevicesDerivedfromBioengineered Plants for Use in Humansand Animals (2002) • ICH Q7 - Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients • ICH Q5 series – Quality of BiotechnologicalProducts • EMEA – Guideline on the Quality of Biological Active Substances Produced by Stable Transgene Expression in Higher Plants (2008) • Guideline on Good Agricultural and Collection Practice (GACP)

9. QualityRequirements for Plant DerivedBiologics • Meet ‘the samequality standards as drugsproducedby conventionalplatforms’ • ‘PMF-deriveddrugs are subject to the samegeneralsubmissionrequirements…as otherbiologicaldrugs’ • Address ‘risks unique to PMF production platforms, particularly in the pre-harvest and harvest stages …’ • Concern about control of plants in a field

11. Stock Maintenance • All startingmaterial must bedefined • Origin and maintenance • Agrobacteriumstock • Managedthrough the use of Master and WorkingCellBanks • Based on principlesdefined in ICHQ5 • Plant seed stock maintenance • Nicotianabenthamiana • Self germinating - homogeneity • Propogatedduringwinter in Quebec (to preventforeign pollen introduction) • ParenteralSeed Stock • Regular (5 year) regenerationwith cumulative stabilitybeingmonitored • AnnualSeed Stock • Producedannuallyfrom parental stock • Aliquoted and stored in independent locations

12. Quality System (‘GMP-like’ controls) – to produce a well-definedbiologicalstartingmaterial • Staff qualification and training • Writtenprocedures • Incomingmaterialspecification • Batch records • Biologicalmaterialspecifications for plants and harvestedmaterial • Trending, including monitoring of production consistency, environment • Characterization such as fiber content, Mycotoxins, Mold + yeast, plant virus, bacterial flora, substrate pH, substrate conductivity • Identifying key control parameters • Product safety first • Parametersthat have an impact on productattributes and yield • Othertraditionaloversight - RiskAssessment, Audits, Change Control • Equipment controls

13. Greenhouse (Upstream) • Controlledprocessthatresults in a definedbiologicalstartingmaterial • The greenhouse environment is designated as a “controlled, not classified” environment • Quality system with QA and QC components • ‘GMP-like’ principles • Not all pharmaceutical GMP regulationscanapply

14. Production of Plants

15. Medicago’s Controls on Plants • All operations covered by production batch records for generation of the critical raw material • No herbicides, pesticides or fungicides • Specifications for input raw materials • Peat, fertilizer, water • Control of entry • Personnel, filtered air (not HEPA), insect monitoring, rodent exclusion and monitoring • Criteria for acceptance as plants proceed from seeding to infiltration • Routine checks for plant viruses and organisms that could produce mycotoxins • No documented cases of human viruses infecting and replicating in plants or plant viruses replicating in humans

16. Plant Control • Controls are in place to assure plants provide proper protein expression • The “working operating space” for many variables has been demonstrated to be large which makes for a robust production system • 20 to 50 gm plants have been used to produce the influenza vaccine with no negative impact to yield or CQA • Other variables have more impact on protein expression • Flower stage is important to control • The plant has been mapped to understand which tissues produce the most product • Kinetics of expression are determined for each strain

17. Plant Certificate of Analysis

18. Downstream • After extraction – purification issimilar to otherbiologicalproducts • Plant specificconcerns – real or perceived • Impuritiesfrom plant manufacturingprocess • Fibres, lipids, host cellproteins • Plant secondarymetabolites • Riskassessment • Consider as well the potentialallergenicpotential • Definetheirnature • Reduce to safelevels • Plant glycans (part of the glycoproteins) • Potentialdiffierences in glycan structure • Analysis and riskassessment • No evidencethey are immunogenic • Couldbebeneficial to processcomparedwithmammaliancellglycans • Product and use specific (vaccine vs long termtherapeutic)

19. Process Validation (PPQ) • Master validation plan outlines methods • Data mining performed on 3 production process generations • Data from > 20 than runs used to set CPP ranges based on CQA of vaccine • Secondary dataset for current process compared back to original data mine • These data will be used to set the acceptable ranges for the consistency lots used for Phase 3 manufacture • Agreement on proposal to the Agency for consistency lots • Obtained buy in for number of lots per strain and number of runs to generate drug substance • Three fills planned with CMO to validate filling process • Matrixed based on number of units filled

20. CPPs for the Production Process • 5 CPP around filtration operations • 3 CPP around chromatography • 1 CPP for virus inactivation • Quality mandated CPP for filtration (2) and virus inactivation (1) • for example – Sterility • Moving forward some CPP may be eliminated by incorporating PAT or reducing risk

21. Containment – Material and Process • Clarification and downstream operations are similar to traditional bioprocessing • Use of disposables • Where stainless steel is used, cleaning validation is traditional • Look for plant based compounds (nicotine, chlorophyll, phenolics) • Closed systems as we approach drug substance • Single use bag technology • VLPs pass through a 0.2 µ filter • Facility design utilizes typical classifications • Greenhouse and VLP (or protein) extraction are the areas that are dissimilar to traditional bioreactor based biopharmaceutical operations

22. Process Challenges from a “Contaminant” Perspective • A greenhouse is not an aseptic environment • Efficient operations require recycling of materials • Water, fertilizer • Biological raw materials • No animal derived components • Many not be “low bioburden or sterile” • Agrobacterium is Gram negative • Endotoxin (but of low pyrogenicity) • Human interaction in an open system with critical raw materials • Plants • A VLP is much like a virus from a surface biochemical point of view • Hard to get a process to have high levels of viral removal/inactivation

24. Bioburden Clearance in the Process • Centrifugation and microfiltration early in the process reduce bioburden • Sterilizing grade filter removes Agrobacteriumearly in the process and reduces all bioburden prior to process intermediate entrance into downstream operations • Additional microfiltration protects chromatography columns and reduces any bioburden load • Sterile filtration prior to fill of bulk drug substance and again prior to fill to syringes or vials

25. Viral Risk • Plants are considered as a low risk for introduction of adventitious agents • Human viruses have not been shown to replicate in plants • Plant viruses are not known to infect human cells • Plants act as a barrier to virus introduction into the process • No animal derived components used in the process • Largest risk is introduction of an adventitious agent into the process by human, rodent or insect. • Regulators worry about new threats emerging based upon past experience in biological processes • Plant based production system presents a low risk of AA introduction when the proper controls are in place

26. Viral Clearance • Many negotiations with the Agency to move from safety testing to inactivation • Inclusion of a UVC treatment unit operation in the process • Development work performed to identify the optimal dose to inactivate a variety of viruses with minimal impact to the product • Added bonus of spiroplasma and mycoplasma inactivation

27. UVC Development • 5 Viruses used to assess system effectiveness • Wide range of doses tested • Looked at inactivation efficiency and impact to product • Data mining from process to assess control limits and risk

28. UVC Viral Inactivation • Process validation completed • Scaled down model equivalent to manufacturing process • Rationale documented to support chosen studies (Risk based) • Used the development data to set boundries for validation study • Duplicate runs with three viruses using two HA types at a single UVC dose • Robustness studies to assess processing and equipment extremes at chosen UVC dose • Re-use of flow path • Regulatory • Negotiation for required steps and clearance levels • Required parallel AA testing for a period to establish safety • Interested in product impact-immunogenicity for each strain

29. Validation: Normal Operating Conditions • MMV spike • B Phuket and H1 California strains • Conclusion: Clearance equivalent to previous • experimental results, complete inactivation

30. Summary • We at Medicago have developed our strategy for containment and control based upon Canadian, ICH and FDA guidancesto the industry • Quality Systems and GMP principles cover the entire process including the plant ‘biological starting material’ • The use of plants as “bioreactors” results in containment that differs from traditional biotech operations • Downstream operations are similar to those used in other biotech processes and result in efficient and timely removal of bacterial microorganisms and host cells • Process validation and QBD principles apply to processes producing biotherapeutics and vaccines from plants as they do to other substrates • While plants may be inherently safe with regard to expansion of human pathogens, the regulatory agencies have concerns with raw materials and vectors that may introduce adventitious agents into plant derived products