Manufacturing of Plasmids for Gene Therapies

Manufacturing Plasmids for Gene Therapies

Paul Lloyd-Evans PhD NHS Blood and Transplant

• Special Health Authority. Over 5000 in work force

• Located over several sites in England. HQ Watford & Filton

• Provide a blood and transplantation service to the NHS, looking after blood donation services in England and transplant services across the UK. • Blood Services Blood & platelet donation Manufacturing of blood products for transfusion

• Transplantation Services (includes donation, processing) Organs, tissues and eye Bone Marrow, Cord blood CD34+ stem cells, DLI’s, T Reg’s

• Diagnostic Services (in support of transplant & transfusion)

• Therapeutic apheresis Services

• R&D / Clinical Trials Cellular & Molecular Therapies Function • Function formed 2013 • 7 HTA and 3 MHRA licensed facilities • Supports 1,400 BM Transplants per annum

DNA-based therapies Protein-based Cell-based therapies CBC therapies CBC 3 x ATU Clinical Biotechnology Centre Historical Milestones

• Facility opened April 1999 • First Recombinant Mab released for Clinical Trial 2001 • First plasmid DNA released for Clinical Trial 2002 • MHRA Manufacturing Authorisation 2004 • 1st Commercial Contract 2007 • Variation to MHRA licence to Import IMP’s 2008 • Collaborator on 2010 £1.2 million EME NIHR grant • Strategic alignment with UCL Gene Therapy Consortium • Part of the Cell and Molecular Therapies Function within NHSBT 2013 • Business case for expanding and relocating facility 2016 • Concept Design completed 2017 Products & Capacity

Products

• Plasmid DNA (< 3 g)

• Recombinant Proteins (< 3 g)

• Importation of ATIMP’s from 3rd Party Countries

Capacity

• Able to perform 2 products in parallel on a campaign basis

• 22 plasmid DNA slots per year dependent on scale of manufacturing Gene Therapy

‘Process of introducing genes into a person’s cells to fight or prevent disease’

Initial focus on monogenetic disorders but now cancer

All use DNA but either presented or packaged differently

Gene Transfer

Viral Vectors Non-Viral

Retro Adeno AAV HSV VLP DNA RNA EP GGun Receptor Lipo

Physical Chemical

Gene Editing can be utilise both Oligonucleotides Naked plasmid DNA Complexed plasmid DNA Advanced Therapies

DNA vector DNA vector

naked DNA vaccination

Stem cells/ immune cells isolated in ATU facilities

Leukaemia, tumours, viruses Clinical Trials

2016 Total of 2210 Gene Therapy Trials

Naked / pDNA 397 Trials - 17%

Adeno, AAV & Retro main viral vectors

Plasmid required as starting material

pDNA Structure

• Double stranded DNA Origin of Replication Promoter • Derived from E.coli Resistance gene Plasmid • Engineered to introduce gene(s) Cloning 3 - 25 kb Site & Gene • Protein expressed from inserted gene Immunostimulatory PolyA • Manufactured in E.coli by replication site

• Excellent safety profile

• Exist in different forms (Topology)

Supercoiled Open circular Advances in construction

1. Improved Immunostimulatory elements for vaccines IS Biological Molecules :Cytokines, Chemokines, CD40L, HSP, Complement Toxins e.g. Tetanus Toxin – use Domain 1 or 2 of Frag C to stimulate MHC Class I or II

2. Improved Safety for vaccines Removal of unwanted bacterial elements : Removal CpG motifs that cause gene silencing, Promotor and antibiotic genes Use of Balanced lethal genes for stability of plasmid Minicircle DNA – intramolecular recombination in cell line to excise unwanted elements

3. Improved plasmids for Viral Vector construction Increased safety - reduce replication competency Reduce number plasmids required for production, better targeting of virus, increase titers DNA Vaccines

• In 1990, University of Wisconsin, Jon Wolff found that injection of DNA plasmids produce a protein response in mice • In 1993, Merck Research Laboratories, Dr Margaret Liu found that intramuscular injection of DNA from influenzae virus into mice produced complete immune response • In 1996, trials involving T-cell lymphoma, influenzae herpes simplex virus were started • In 1999 University of Southampton, Prof Terry Hamblin used 1st DNA vaccine in humans for lymphoma. • Currently 2 Commercial Veterinary vaccines • 1 vaccine undergoing MA in Japan for angiogenesis pDNA as Starting Materials

• Require multiple plasmids for applications • Same manufacturing process as for vaccines • Same testing requirements for vaccines • HQ vs GMP quality • GMP will be required for marketed ATMP’s • Scale of production < 50 mg to > 10 g required • Increased quality demands throughout ATMP development • Increased demand from sector as it grows • Shortage of capacity / parallel production required • Critical in supply chain GMP Manufacture Cell Line Considerations

• Product Quality : Plasmid DNA yields Plasmid DNA Topology Plasmid Stability (viral vector applications) Downstream Processing and impurities Growth characteristics

• Safety : Must conform to EU Biosafety Regulations

• Licensing : Avoid restriction of use, patents rights & infringements Manufacturing Technology

Carnes, A et al 2007 Plasmid Manufacturing Technology in Recent Patents on Technology

Ferment Extract Purification Formulation

Start with the end in mind - QbD

Regulatory Guidelines:

Ph.Eur : Monograph for Manufacture of Plasmid DNA Vaccines for Gene Therapy Ph.Eur : Monograph Products from Recombinant DNA Technology ICH Q2 Validation of Analytical Procedures ICH Q5 Quality of Biotechnology Products Transformation and MCB • Heat shock with plasmid DNA • Grow in presence of selective marker • Clone selection • Lay down MCB / WCB of > 200 vials Fermentation

Goal - Maximise volumetric yield (mg/L) of plasmid DNA and maintain specific yield (mg/g) Why? – economic & size of fermentations

Growth Media

Affects plasmid quality, biomass

Animal-Free – Plant based, no animal enzymes used in their production, TSE risk

Complex, defined or semi defined.

Plasmid Ori (ColE1 / pBR322 / pUC based)

High copy plasmids often have known mutations that affect copy number regulation and upon temp shift in culture can increase pDNA yields

Fermentation Systems :

Stainless predominantly, however Single Use Fermentation Systems being introduced In-process monitoring of pDNA

Important to have rapid, robust, reliable in-process assay for a) Process Monitoring b) Yield estimation and reconciliation c) Assist in Process development

pDNA Calibration Curve on Superose 6 100 µg pDNA Spike Untransformed bacterial lysate 490 y = 1.84x - 2.73 R2 = 0.999

390

290 Ab 260 nm

pDNA Isoforms mAU/ml 190 mAU/ml RNA 90

pDNA -10 0 50 100 150 200 250 300 Plasmid µg/ml

Superose 6 0.5 ml/min 100 µl injection Tris 1.5 M AS Mobile phase

0 0.0 10.0 20.0 30.0 ml ml Extraction

• Extract pDNA without damage. Remove impurities if possible

• Two Methods can be employed:

a) Chemical – detergents, osmotic shock, alkali, enzyme

b) Physio-chemical – shear, ultrasonication, heat, freeze-thaw, Pressure

• Mechanical disruption not used due to unacceptable damage

• Chemical extraction method of choice however variable and volume handling

• Use of enzymes should be avoided

• Challenges for scale up Purification

• Bacterial Lysate complex mixture - pDNA is a minor component < 5%

• Separate and purify molecules with similar chemical /physical properties

RNA All -ve charged HMW & LMW forms gDNA large MW SC/OC pDNA hydrophobic Endotoxin - ve charged

• Remove all impurities to acceptable levels with good yield.

• Plasmid DNA size can influence on both quality and yields

• Develop a platform process

• High Supercoiled content Plasmid DNA Process

Design Activity QC Transformation of Host Cell line with Incoming QC on Host Cell line & pDNA • Small scale batches pDNA In-process QC testing Final QC testing for Prepare Master Cell Bank Identity & Safety Testing • No materials of animal origin to current Regulatory guidelines Upstream Grade D • Disposable Fermentation of cell line In-process QC Harvest cells by centrifugation - • Platform with ease of use Storage of Bulk Harvest - Chemical Lysis of bacterial pellets In-process QC • Low Process Volumes Filtration Step In-process QC

Clarification of lysate by ultrafiltration Hollow Fibre Integrity test • Cost effective Filtration Step In-process QC • Rapid Mustang Q AIEX Capture Step In-process QC

Hydrophobic Interaction In-process QC • Excellent CQA Chromatography Polishing Step Filtration Step In-process QC Downstream Grade C Diafiltration and concentration by In-process QC ultrafiltration Hollow Fibre Integrity test Filtration Step - Drug Bulk Substance In-process QC, Bioburden, endotoxin Filter Integrity test Upstream

Clarified lysate PC056 D 141106001:1_UV2_260nm

mAU 7.35

300

250

200

150

100

21.16 0 19.70

0.0 5.0 10.0 15.0 20.0 25.0 30.0 ml

12 L Fermenter Centrifuge Bacterial Ultrafiltration of Lysate Extraction 10 L

Scale

For > 0.5 g then sub-batch Production Time = Up to 6 weeks

Clarified Lysate < 3 L Downstream

Anion Exchange HIC Ultrafiltration & Formulation Chromatography

Scale

For > 0.5 g Production Time = 2 weeks Bulk Substance < 1 L Fill and Finish

Envair Grade A Positive Pressure Isolator Aseptic Technologies CrystalTM M1 closed vial filling station Bioquell Clarus L3 VPHP generator Aseptic Technologies CrystalTM M1 filling system Aseptic manual batch fill of up to 500 bottles

Bioquell Clarus L-3

1 – 50 ml CrystalTM vial

Closed Vial Technology Process Recovery & Yield

Amount of % Recovery % Yield Step Volume in L pDNA mg over step

25 L 1100 100 100 Fermentation (800 g bacteria)

Clarified Lysate 4.6 L 864 78 78 Mustang Q Capture 2 L 599 69 54 HIC Polish 4 L 586 98 53 Final DF/Conc 0.145 L 480 82 43 BDS 0.13 L 360 92 32

Target of 225 mg of pDNA for patient specific pDNA vaccine – min 60 vials

84 vials filled for FDP

Release of product within 12 weeks QC Analytical Testing

Purity: Content: UV Spectroscopy Purity: UV Spectroscopy Homogeneity: AIEX-HPLC Host cell DNA: Q-PCR Host cell Proteins: Micro-BCA Host cell RNA: RibogreenTM Assay Endotoxin: LAL Assay

Identity: Restriction Digest Fingerprint DNA Sequencing

Adventitious Agents: Bioburden, Sterility

Physio/chemical: pH, Osmolality

Potency: Bioassay (Client) Performance CQA

Table summarising CQA for process performance

Batch SC OD260/280 Residual Residual Residual Residual Number Content Purity RNA gDNA Protein Endotoxin >80 % 1.7-2.0 µg/mg µg/mg µg/mg EU/mg PB065 98 1.9 7.2 1.3 2.5 <10 PB065 95 1.9 21.7 1.35 1.4 <10 PB066 93 1.9 20.8 8.2 2.7 <10 PB066 95 2 8.4 6.8 0.7 <10 PB069 92 1.9 10.9 1.75 <0.8 0.6 PB070 95 1.9 14.3 1.2 0.32 0.1 PB072 93 1.9 16.4 2.36 <1.45 <0.02 PB073 92 1.9 8.1 3.09 <1.6 0.213 PB074 98 1.9 12.1 0.31 <0.8 1.4 PB078 94.5 1.9 9.7 0.42 <1.6 0.082 PB079 94 1.9 16.3 0.47 <1.6 0.144 PB080 97.6 1.9 6.2 0.34 <1.6 0.147 PB084 94 1.9 13.9 3.37 3.01 <0.01 PB087 94.9 1.9 8.8 2.54 2.9 0.085 PB125 94.3 1.9 9.2 1.16 1 0.394

AVERAGE 94.7 1.9 12.5 2.3 1.9 0.4 SD 1.9 0.03 4.9 2.3 1.1 0.4

Excellent Critical Quality Attribute Profile

Yield approx 30% Stability Studies

• Stability Indicating parameter TSK DNA-NPR Column TosoH

060907 50pc 001:1_UV1_260nm 060907 50pc 001:1_Conc 060907 50pc 001:1_EditedBaseline

• Validated suitable assays mAU 8.61 9.86 150 • ICH 2 Guidelines

100 • Real time or Accelerated Studies

Time Points in Months Tests 3 6 9 12 18 24 0.0 5.0 10.0 15.0 20.0 ml Appearance X X X X X X DNA Conc X X X X X X SC Composition X X X X X X Restriction Mapping X X X X X X Sterility - - - - - X Manufacture 2015 -17

Product and indication Customer Plasmid DNA for the generation of iPSC for cell therapy UCL treatment of acute macular degeneration

Plasmid DNA for the production of GMP - grade iPSC cell lines Commercial

DNA vaccines for immunotherapy of HIV Academic & Commercial

Plasmid DNA for immunotherapy in breast cancer Commercial

Plasmid DNA for viral vector construct for treatment of UCL & Commercial Parkinson’s disease Plasmid DNA for viral vector construct for treatment of UCL & Commercial Retinopathies (various)

Plasmid DNA for viral vector construct for treatment of UCL Tay Sachs Syndrome

Plasmid DNA for viral vector construct for treatment of UCL & Commercial Haemophilia

Plasmid DNA for viral vector for SCID Commercial

Over 15 Clinical trials and over 400 patients treated since 2004 Looking to the future…

• Strategic intent of NHSBT is to support novel regenerative medicine therapies • Develop the facilities with cutting edge technology to meet these challenges • Harness and capitalise on synergies within organisation and BTRU’s • Develop and bring through own products • Double manufacturing capacity for DNA based products • Scale up to up to 5 g pDNA batches • Increase QC capacity and capabilities for DNA based products • Diversify product range • Continue to train and develop staff with necessary skills Acknowledgements

Dr Jon Smythe Head of CMT

Quality Control Becky Clare Head of QC Adam Ziegler QC Supervisor Jillian Harris QC Supervisor Amanda Keyes QC Scientist Emma Taylor QC Scientist

Development Function Dr Piers Walser Lead Development Scientist Antony Wright Senior Development Scientist Laura Barry Development Scientist

Production Denise Phillips Head of Production Helena Pastor Production Supervisor Tracy Hutcherson Production & Development Scientist Sujata Rai Production Scientist Claire Witcombe Production Scientist

QA / QP Function Tracey Miller QA Assistant Manager Fred Goddard QA Manager Keith Smith Qualified Person Karen Burks Qualified Person Back up slides Process Flow

Business Receipt of Incoming GMO Risk Batch Record Development Quotation Contract Development material QC Assessment Compilation Activities

5 days 30 days 2 days 1 day 1 day 5 -10 days

BMR Label Store Product Batch Record Production Fill Product QC Analysis Issue C of A Pre-approval Product in Quarantine Review

32.5 days 2.5 days 30 days 1 day

Store Product Stability Archive Shipment Invoice in Issue Trials Records Total Time: < 120 days Production time : 35 days QC & Release: 32 days 0.5 day 0.5 day Product Support:: Ongoing New Process Technology

Anion Exchange HIC Chromatography

Ultrafiltration of Lysate

30 - 50 L Fermenter Scale

For 5 g Production Time = Upstream 2 weeks = Downstream 2 weeks