Unique Challenges Associated with the Produc6on of Viral Glycoproteins

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1 Dale and Betty Bumpers Vaccine Research Center National Institute of Allergy and Infectious Diseases National Institutes of Health Department of Health and Human Services Unique Challenges Associated with the Produc6on of Viral Glycoproteins Richard M. Schwartz, Ph.D. Vaccine Production Program Vaccine Research Center June 11, 2015 HIV ENV Manufacturing Workshop

2 Outline Strategies for Lot Release Assays Challenges in Cell Line Development Challenges in Env Purifica6on Choices for GMP ENV Upstream Produc6on Example of ENV produc6on using RSV F trimer as a model system

3 Required Lot Release Assays Potency Content (Strength) ID Purity (Impurities) Safety 3

4 RSV Trimer Assays for Development Lot Release Assays ALribute Assay Alterna6ve Potency Octet ELISA, In vivo Content HPLC BCA ID (per ENV) cief, WB, HPLC Digest/HPLC Purity GXII SDS Gel Characteriza2on Assays ALribute ParFcle/protein Size ParFcle Structure Host Cell DNA QuanFty and Size Residual Benzonase AcFvity Residual mab impurity (if affinity separafon) QuanFtaFon of Host Cell Protein Assay DLS TEM qpcr kit Cygnus Kit Cygnus Kit 4

5 Select an In Vitro Potency Assay Criteria During Research Phase Determine Critical Epitope(s) for ENV activity/ antigenicity Select and measure bnab(s) for critical epitope(s) in early non-clinical research materials Don t assume that all bnab epitopes are stable bnab Binding May be the Optimal Potency Assay bnab binding needed for product quality assessment This will be the workhorse assay for clone selection and titer measurement May want to use 1 bnab for potency and others for characterization Potency spec is developed using research material through multiple development batches 5

6 Develop CQAs and ID Assay Early in Development Orthogonal methods are developed to target CQAs Octet/Biacore for epitope(s) availability and affinity SEC/DLS/GXII for trimer/protein size Glycan analysis may be critical Use CQA assay(s) throughout the manufacturing process to measure product quality Consider Identity assay development early if more than 1 ENV is planned for manufacturing Need to be able to differentiate ALL ENVs from each other for lot release May not be possible using only bnabs The simpler the assay the better 6

7 Biolayer Interferometry (Octet) for Potency Determination 1) Load sensor anti-env bnab 2) Capture ENV 3) Signal of ENV Captured concentration of active epitope

8 RSV Octet Cell Culture Titer (Potency) Assay 5C4 D25 AM14 Synagis 8

9 CELL LINE DEVELOPMENT CHALLENGES

10 Choice of Cell Host Line Cri6cal CHO is typical cell line of choice Technology for stable CLD and producfon readily available Requires viral clearance ENV must be stable at low ph or in detergent CriFcal to study low ph stability early in research phase Endogenous furin or other enzymes required for Env cleavage may not be sufficient

11 Example: RSV Trimer Cell Line Development Stable expression of DS- Cav1 in CHO DG44 Cells vectors with DHFR selecfon vector with and without furin DGV vector with F and furin Stable expression of DS- Cav1 in HEK- 293 cells

12 RSV- F Cell Line Development in CHO Cells I. 2.1 RSV- F DHFR Vector MTX Selection 48h Post Transfection II. 2.1 RSV- F + Furin DHFR Vector Early Stable Pools III. 2.1 RSV- F + Furin DGV DHFR Vector CDM4CHO Medium IFA I, II & III I, II & III TC-96W TC-24W TC-6W D 9-11D D CDM4CHO + 50 nm MTX CDM4CHO + 50 nm MTX CDM4CHO + 50 nm MTX Semi-solid medium With 50 nm MTX Amritha Menon/ Mingzhong Chen Motavizumab dot blot based Clone Screening 12

13 RSV Cell Line Development Picking Clones Typical CLD uses FACS or ClonePix for clone selec6on ClonePix and FACS require GMP- grade mab reagent or use random clone picking Cri6cal to develop early analy6cs to screen producing from non- producing colonies Need to define crifcal ENV epitopes and have mab(s) for clone selecfon (use the potency assay if available) mabs to mulfple epitopes may be useful to define intact ENV protein

14 Which mab is Best for Clone Selec6on? RSV HIV CD4 V1/V2 MPER? McLellan et al. Science 2013

15 Screening for RSV F expressing CHO Cells Using Dot blot - Motavizumab posi6ve isolates No GMP-grade mab for Clonepix or FACS 3000 colonies screened for RSVF by dot blot RSV-F dot blot positive isolates Positive control DS-Cav1 15

16 DS- Cav1 CLD - Low Produc6vity in CHO DG44 Cells 96W plates (D10) T o p C lo n e s A B C u g /m L B E F G 2 0 A M C 4 D 2 5 S y n a g is M o ta v iz u m a b A n ti R S V m A b

17 DS- Cav1: Low Transient Produc6vity in CHO Cells Compared to HEK- 293 Cells A M 1 4 (2 9 3 ) 5 C 4 (2 9 3 ) 6 0 * * * 6 0 * * * m g /m L m g /m L D 1 D 2 D 3 D 4 D a y s P o s t T ra n s fe c tio n D 5 S y n a g is (2 9 3 ) 0 D 1 D 2 D 3 D 4 D 5 D a y s P o s t T ra n s fe c tio n 6 0 m g /m L HEK D s C a293 v 1 Tagged CHO D G V HEK D s c a N o No-tag T a g * Assay saturation 0 D 1 D 2 D 3 D 4 D 5 D a y s P o s t T ra n s fe c tio n

18 Confirma6on of Low Expression in CHO DG44 Cells 293 expression Vectors for transient expression of DS- Cav1 in DG44 cells A M 1 4 (D G 4 4 ) 5 C 4 (D G 4 4 ) m g /m L m g /m L D 1 D 2 D 3 D 4 0 D 1 D 2 D 3 D 4 D a y s P o s t T ra n s fe c tio n D a y s P o s t T ra n s fe c tio n S y n a g is (D G 4 4 ) D s C a v 1 m g /m L D G V D 1 D 2 D 3 D 4 D a y s P o s t T ra n s fe c tio n

19 New RSV F stable Pool CAG promoter into 4.0 backbone SV40 promoter replaced by CAG promoter

20 DS- Cav1 CLD in VPPL CHO- DG44 Host Cells CHO DG44 transfected cells seeded in low glutamine containing semi- solid White based colonies picked using ClonePix Significant increase in percent posifve colonies observed compared earlier CLD campaign Dot blot posifve clones moved from 96-24W- SF Synagis 5C4 AM14 DSCAV1 EXPI D5 SUPE 4.1 F 4.1 F & Furin 2.1 DGV 4.1 F 4.1 F & Furin 2.1 DGV 4.1 F 4.1 F & Furin 2.1 DGV New Expression vectors F F + Furin DGV CHO-DG44 ClonePix Clones in SF Transfection

21 Improved Yield in CHO DG44 Cells - Low glutamine and improved MTX selec6on in semi- solid medium 3 0 D G (F + F u r in ) C lo n e s (L G ) 2 0 m g /m L C 4 A M 1 4 D 2 5 R S V m A b DG44 colonies picked at low glutamine in semi- solid medium by octet show ~ 5- fold increase in Fter compared to earlier clones

22 VRC293 and CHO DG44 Clones Expressing DS- Cav1 V R C E a r ly P a s s a g e m g /m L C 4 D 2 5 A M C 4 D 2 5 A M 1 4 R S V m A b

23 UPSTREAM PROCESS OPTIONS

24 HIV ENV Manufacturing Upstream Overview Transient protein producfon More rapid development of mulfple ENVs Much more labor intensive upstream than stable CLD More variable producfon Fter and possibly quality Stable cell line producfon Long lead Fme for CLD Less labor intensive cell culture Higher Fter, scalable and more consistent quality

25 HEK- 293 Transient Transfec6on Produc6on Flow: (50L Limit) Vial of Cells 250mL (3-4 days) 1000 ml (3-4 days) 3000 ml (3-4 days) pdna/pei 50L WAVE Bioreactor For transfection/ expression ksep400 Semi-continuous Centrifuge 50L SUB (3-4 days)

26 Stable Cell: Large- Scale Produc6on Flow Vial of Cells 250mL (3-4 days) 1000 ml (3-4 days) 3000 ml (3-4 days) 2000L Fed batch Bioreactor (~14 days) 250L SUB (3-4 days) 50L SUB (3-4 days) 20L WAVE Bioreactor (3-4 days)

27 DOWNSTREAM PROCESS OPTIONS

28 Chromatographic Purifica6on Challenges Method Advantage Disadvantage AEX / CEX Lec6n Readily available Inexpensive Large choice of resins Wide ph range Lower purif factor Be_er purificafon factor Poor regenerafon Poor storage Expensive Poor availability mab Affinity High purificafon factor Need GMP mab Expensive Need mab release & removal Verify mab removal Unknown regenerafon stability Poor storage Expensive Variable conjugafon

29 Viral Clearance for CHO CHO cells require viral clearance Requires >6 logs viral clearance than in the harvest fluid Model enveloped and non- enveloped tested for clearance 2 viral clearance- specific purificafon steps Low ph treatment (ph 3.5 for 1 hour) or Detergent treatment 20nm viral filtrafon 2 other process steps tested for viral clearance Ion exchange chromatography Affinity chromatography

30 Dale and Betty Bumpers Vaccine Research Center National Institute of Allergy and Infectious Diseases National Institutes of Health Department of Health and Human Services VPPL Acknowledgements VCMP (Leidos) John Gilly MaL Westerman Barb Brooks ScoL Emerick Doug Cooper Pat Marshal And many more VRC Labs of: Gary Nabel John Mascola Barney Graham Peter Kwong Nancy Sullivan Srini Rao Julie Ledgerwood