gmp production of a polyvalent recombinant gp120 … · elisa, western blotting rt -pcr + pcr/ qpcr...

GMP Production of A Polyvalent Recombinant

gp120 Protein Formulation:

Cell Line Development and Downstream Purification

Shan Lu, MD, PhD, MHA

University of Mass. Med School, Worcester, USA

HIV Env Manufacturing Workshop

Bethesda, June 11, 2015

UMMS IPCAVD program

• The objective of UMMS IPCAVD is to produce a polyvalent DNA prime-protein boost HIV vaccine

• Initially five primary gp120 immunogens were identified

• Finally four highly purified recombinant gp120 proteins were produced under GMP manufacturing condition

Challenges facing the GMP production of HIV-1 gp120

proteins from a CHO expression system

• Lack of exiting CMO with significant experience – Neglected need for subunit Env-based HIV vaccines – No available qualified CMOs

• Only one with active GMP Env production (Europe based, gp140 only, no in-house cell line experience, high cost)

– CHO cell line requirement • No certified CHO (no prion exposure) available at most CMOs • No CMOs have in-house experience • Dhfr- (MTX use) a potential regulatory concern

– Lack of supporting reagents • For QC and other steps

– Cost • Overall high • Not feasible for scale up production/developing countries

Overall Strategy for UMMS Team

• Formed a special task force to search for qualified candidate CMO

• Conducted an extensive industry-wide global search on protein CMOs

– GMP capability

– Experience with mammalian expression systems

• 14 CMOs were identified and contacted, CDA executed, 10 submitted quotes, 1 had GMP audit and another one informal audit

• Divide the whole process into two components

• Life Technology/Invitrogen PD-Direct service for

cell line development:

– MTX-free, serum free culture medium

– High-producing cell line screening platform

– Stability selection

– Providing qualified cell line – DG 44

• Waisman Biomanufacturing (WB) for GMP manufacturing

– See Part II of this presentation

Life Technologies

Part I: cell line development (Life Tech)

gp120 identification in CHO cell line

Detection level

PCR, sequencing and/or qPCR

DNA

mRNA

protein

Exogenous genes on host

chromosome

ELISA, Western Blotting

RT-PCR + PCR/qPCR

Individual gp120 gene insert cloned in Invitrogen

Topo vector was transfected into DG44 CHO cells

gp120 CHO Cell Development Overview (sample)

Action Location Status

Prepare 5 gp120 transfected clone pools LT Complete

Primary pools screening for gp120 titer UMMS Complete

Carry out ClonePix Operation to identify highly

expressing clones x 5 LT Complete

Growth and Expansion of > 96 x 5 Clones Post Picking LT Complete

Secondary Clone Screening for Protein Titer UMMS Complete

Expansion of Clones and Freezing of Top Leading

Clones 10 x 5 clones LT Complete

Tertiary Screen of Top 10 Leading Clones x 5 in shake

flasks LT Complete

Tertiary Clone Screening for Protein Titer UMMS Complete

Shake flask stability thru 60 generations LT Complete

Stability Clone Screening for Protein Titer UMMS Complete

Decide on lead clone and top back up clones x 5 LT/ UMMS Complete

Cell identity test for the top clones UMMS Complete

Clone bank testing x 5 (e.g. sterility, Mycoplasma) LT/subcontr Complete

Deliver Top Clone x 5 LT Complete

Step 1: Transfection and Selection

-8.33E-17

0.03

0.06

0.09

0.12

0.15

13 15 11 19 111113

O.D

. 45

0/6

20

Sample #

UM 1 A

1:2

1:10 00.030.060.090.120.15

23 25 27 29 211 213

O.D

. 45

0/6

20

Sample #

UM 2 A

1:2

1:100.0000

0.0300

0.0600

0.0900

0.1200

0.1500

33 35 37 39 311 313

O.D

. 45

0/6

20

Sample #

UM 3 B

1:2

1:10

0

0.03

0.06

0.09

0.12

0.15

43 45 47 49 411 413

O.D

. 45

0/6

20

Sample #

UM 4 B

1:2

1:100.0000

0.0300

0.0600

0.0900

0.1200

0.1500

53 55 57 59 511 513

O.D

. 45

0/6

20

Sample #

UM 5 C2

1:2

1:100.0000

0.0300

0.0600

0.0900

0.1200

0.1500

63 65 67 69 611 613O

.D. 4

50

/620

Sample #

UM 6 C2

1:2

1:10

0.0000

0.0300

0.0600

0.0900

0.1200

0.1500

73 75 77 79 711 713

O.D

. 45

0/6

20

Sample #

UM-7 AE

1:2

1:10

0.0000

0.0300

0.0600

0.0900

0.1200

0.1500

93 95 97 99 911 913

O.D

. 45

0/6

20

Sample #

UM 9 D

1:2

1:10

0.0000

0.0300

0.0600

0.0900

0.1200

0.1500

83 85 87 89 811 813

O.D

. 45

0/6

20

Sample #

UM 8 AE

1:2

1:10

0.00000.03000.06000.09000.12000.1500

O.D

. 45

0/6

20

Sample #

UM 10 D

1:2

1:10

Transfection supernatant: gp120 Detecting rabbit sera Rabbit study 130, pooled bleed VIII (rabbit #795-799) Sera dilution 1:250

UMMS ELISA test on the expression of gp120 from Life Tech

Step 2: Amplification

Step 3: ClonePix Feasibility

Step 4: Single Cell Cloning

Top 10 samples UM2-1000

# sample Conc. (µg/ml)

2E10 41.30

4F2 45.19

3B1 47.08

3D1 52.52

2E6 63.80

4D7 34.30

4A10 36.96

4G6 54.90

2C5 35.27

2H5 48.36

The concentrations determined here are generated from the repeated measurements of the top 24 samples.

3

Western Blot with gp120s

from the top 10 clones of UM7-1000

M 1B10 1D10 1E9 2A1 2D2 AE (+) CHO M 2G10 2H9 3D6 3H5 3F6 AE (+) CHO

175KD 175KD

80KD 80KD

Tertiary Screen

Top 10 samples UM2-1000 (P1)

KW SW SW SW SW SW KW KW KW KW

V V

V V

Two of the ten clones (2C5, 4D&) expressing P1 show high doubling time. The remaining grow satisfactorily.

UMMS LifeTechnology secondary tertiary

# sample day7(2nd) day7(3rd) average(7-14) average(12d)

2C5 35.27 58.98 113.59 82.83

2E10 41.3 219.23 190.24 143.90

2E6 63.8 131.08 239.68 184.74

2H5 48.36 90.28 112.57 81.85

3B1 47.08 221.29 186.82 135.06

3D1 52.52 218.47 238.39 173.49

4A10 36.96 61.89 68.33 52.35

4D7 34.3 37.46 82.28 60.85

4F2 45.19 86.63 102.13 73.93

4G6 54.9 110.30 145.26 107.21

0

50

100

150

200

250

300

2C5 2E10 2E6 2H5 3B1 3D1 4A10 4D7 4F2 4G6

day7(2nd)

day7(3rd)

average(7-14)

average(12d)

Step 5: Stability

0

50

100

150

200

250

300

350

400

450

500

4G6* 4F2 4D7 4A10 3D1 3B1* 2H5 2E6 2E10 2C5

ave

rage

pro

tein

co

nce

ntr

atio

n

clone

UM2-1000 Stability

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

50L Bioreactor pilot run with clone A: Levels of gp120 protein production

0

50

100

150

200

250

300

350

Day0 Day1 Day2 Day3 Day4 Day5 Day6 Day7 Day8 Day9 Day10 Day11 Day12 Day13 Day14

Sandwich ELISA with RmAb76/16-HRP

Summary of Part I

• Used ClonePix to identify high expression CHO cell colonies

• UMMS group provided rabbit mAb reagents for ClonePix

• UMMS group established several unique capture ELISA kits to identify and quantify gp120 from very different subtypes

• The whole process included multiple rounds of selections and a stability test

Case 2 Improve the purity of recombinant gp120 proteins CMO: Waisman Biomanufacturing University of Wisconsin - Madison

Director: Derek Hei, PhD

Waisman-UMMS Recombinant gp120 protein Program Overview

• Cell bank production/testing/release – 5 CHO MCBs

• Cell culture development – feed strategy up to 50L

• Downstream purification development

• Analytical method development and qualification

• Development of cGMP documentation

• Tox/Qual Lot Production –3L and 50 L pilot and engineer runs o Reference standards

o Stability study

o Viral clearance studies

o Materials for Tox study

• Clinical production – planned for 2000 doses/lot

• Aseptic fill/finish

• Pre-IND and IND documentation support

Options for HIV-1 Env Purification

• Approaches based on affinity purification

o Lectin:

o UMMS-ABL DP6-001 program

o Env-specific mAb:

o European HIV vaccine program (gp140 site)

o Tagged protein: His or other tags (mainly pre-clinical studies)

• Non-affinity based approaches

o Preferred by major industry players

o Consistency

o Scale-up potential

o No contamination of reagents included in affinity approach

o Avoiding cost and GMP production of affinity process reagents

o Challenges

o Unique chemical nature of HIV-1 Env (glycosylation)

o Hard to separate targeted Env species from impurities

Process Development Results presented at July 2013 SAB

50L SUB

Clarification (Depth Filtration)

UF/DF (TFF1)

Anion Exchange Chromatography

gp120 in FT

Cation Exchange Chromatography

gp120 Bind/Elute

UF/DF (TFF3)

Size Exclusion Chromatography

Original gp120 Purification Process Flow Diagram (presented at July 2013 SAB meeting)

Viral Filtration

= viral clearance step

UF/DF (TFF2) pH 4.0 Precipitation and

Filtration UF/DF (TFF4)

Fill

Key Elements of Downstream Purification Process

• A gp120 protein-specific purification process

o Anion exchange

o Removal of genomic DNA from bioreactor harvest

o gp120 in flow through-some contaminating proteins removed

o Low pH precipitation

o pH drop induces precipitation of contaminating proteins

o Cation exchange

o gp120- bind and elute step

o Size Exclusion Chromatography

o Controlling monomer/dimer ratio

o Removal of smaller MW contaminants

220

160

120 100 90 80 70 60

50

40

30

25 20

15

10

(kD) • Load = gp120B Qual/Tox Lot 1 CEX pool

• 300mL Sephacryl S200 60cm column

• Flow rate = 15cm/hr

• 1% column volume load at 10mg/mL

• Non-Reducing gel-4-12% Bis-Tris/MES

load

Dimer

Monomer

Best results presented at July 2013 SAB meeting: 50L gp120B1 lot, SEC Step

Dimer

Monomer

Gp120 or

Contaminating

Proteins?

Additional degraded bands shown

by reduced SDS-PAGE

220

160

120 100 90 80 70 60

50

40

30

25 20

15

10

(kD)

B12 B8 A8

A0

A10 B10 A6

A1

220

160

120 100 90 80 70 60

50

40

30

25 20

15

10

(kD)

B12 B8 A8 A10 B10 A6

Non-reduced Reduced

A0

A1

Dimer

Monomer

A8

Dimer

Monomer

Gp120 or

Contaminating

Proteins?

Gp120 or

Contaminating

Proteins?

Further tasks to improve the outcome of purification process

Task One

To identify the nature of low molecular bands

Task Two

To remove degraded gp120 and non-gp120 materials

Identification of Clipped gp120 Fragments by N-Terminal Sequencing

Technical Steps

The gp120 proteins were separated under reducing conditions by SDS-PAGE

The gels were stained with Simply Blue and destained

The stained proteins were transferred to PVDF membrane and allowed to dry overnight

The membranes were sent to Larry Dangott at Texas A & M Bands of interest were cut from the membranes and N-terminally sequenced

SVEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVTEHFNMWKNNMVEQMQEDIISLWDQSLKPCVKLTPLCVTLNCKDVNATNTTNDSEGTMERGEIKNCSFNITTSIRDEVQKEYALFYKLDVVPIDNNNTSYRLISCDTSVITQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIHIGPGRAFYTTGEIIGDIRQAHCNISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV

Cleavage site: Arginine 282-Alanine 283

Legend: Identified AA’s by N-terminal sequencing: AA’s V2-L5 and A282-T286 AA S-1Found in N-term. Sequencing, but not in the provided sequence V3 Loop AA’s

N-Terminal sequencing results for reduced gp120B fragments

Observed native N-Terminus (β1-90kD band): S-V-E-K-L Observed cleavage site (β2-70kD band): A-T-G-D-I-I-G

M. N R N R KDa 250

150

100

75

50

37

25 20

15 10

C1 V1 V2 C2 V3 C3 V4 C4 V5 C5

R15 R13

Further confirmed by mapping with gp120-specific rabbit mAbs

M: MW marker (Bio-Rad)

N: Non-reduced

R: reduced

R15

M N R

R53 R13

R53

R16

N R M M N R

R20

50kD 70kD

R16 R20

70kD 50kD

A key finding

• There are two protease cleavage steps

– Cleavage at the stage of cell culture

• V3 based, but with sequence preference of GPGR-

A (subtype B)

– Cleavage at post-harvest (during purification)

• Multiple sites, non-subtype B clones

• What are the responsible enzymes?

The gp120 V3 loop where the N-terminus of the 70KD fragment was observed

V3 loop

RP-HPLC analysis on gp120B1 A7-B11 Peak Collection

(Intentionally Including All Major Contaminants)

min7.5 10 12.5 15 17.5 20 22.5 25

mAU

0

5

10

15

20

25

30

DAD1 D, Sig=280,10 Ref=360,10 (12AUG2013\12AUG2013 2013-08-12 16-08-21\DEF_LC 2013-08-12 14-31-16\001-0101.D)

Area

: 175

4.1

Area

: 540

.805

Area

: 221

.051

Area

: 92.7

224

Area

: 45.0

52

11.

135

13.

569

16.

256

17.

709

18.

365

Peak 1

~67.95% Peak 2

~19.79%

Peak 4

~7.71%

Peak 5

~3.39%

Peak 6

~1.15%

Identification of contaminating proteins

SDS-PAGE Gel image

220

160

120

100 90

80

70

60

50

40

30

25

20

15

10

220

160

120 100

90

80

70

60

50

40

30

25

20

15

10

Peak 1 2 4 5 6

MW MW

Both N-terminal Sequencing and

Mass Spec analysis were conducted

on collected peaks.

Findings:

1. Low MW bands co-eluting with

gp120 at SEC step are non-

gp120 related;

2. Various peaks represented

different CHO-associated

enzymes

3. Low pH degradation was found

at CEX step

Challenges:

1. To remove enzymes

2. To reduce gp120 degradation

5

Lysosomal protective protein*

Tissue alpha- L-fucosidase*

Dipeptidyl- peptidase 2

Strategies to minimize gp120 clipping

and improve the removal of impurities

Strategy One

- upstream cell culture related activities

Optimization with Cell Culture Activities

- Culture conditions:

- Various feeding strategies, and reduced Temp

- Scale of fermentation and control process

- Culture harvest time (day 7-14)

- Finding: processing starts during seed train and continues during bioreactor process

- Freezing harvest

- No reduction of processing/degradation observed in purification process

- Culture additives tested

- Plant hydrolysate, FBS, BSA, AA, Ex-Cyte, ferric citrate, base media, protease inhibitors

- No reduction of processing

Letter to Nature

Immunogen: gp120-IIIB with 25 aa of HSV-2 gD at the N-terminal of gp120 aa 61 (31 after leader)-531

gp120 (aa 61-531) gD (25aa)

VaxGen Chimpanzee Studies

Study 1 (2 animals) Lectin purified gp120 (~50% pure) X4 + anti-gp120 antibody affinity purified gp120 (95% pure) X1 Study 2 (2 animals) Anti-gp120 antibody affinity purified gp120 (95% pure) X 3

Challenge virus: HIV-1 IIIB virus, Route: i.v. Study 1: 100 TCID50, 4 weeks after last immunization Study 2: 10 CID50 (40 TCID50), 3 weeks after last immunization)

Outcome (based on post challenge sero-conversion and PBMC co-cultivation) Study 1: not protected Study 2: protected

Summary of chimp studies

Inconclusive chimp study results ? V3 cleavage related protection

Small sample size

Study One also used 95% pure gp120 in the last immunization

Lower challenge dose in Study Two

IIIB virus is V3 dependent as a TCLA virus, different from primary HIV isolates to be protected in human vaccine development

VRC01 IgCD4 2G12 R53 R28

Waisman CHO gp120 (mixture of process and non-processed monomers)

293F produced gp120 (non-processed monomers)

1:1 binding model

Similar binding with well characterized mAbs

KDa 250

150

100

75

50

37

25 20

15

1. Protein marker 2. gp120B-3L-2 Ref Std (mixed) 3. Purified Intact gp120B 4. Flow-though, gp120B

(mixed) 5. gp120-JRFL (293F) as control Intact gp120B was separated from mixed gp120B by rabbit mAb 28 SDS-PAGE using reducing condition

gp120B-3LSUB Intact Protein Preparation by rabbit mAb R28 affinity purification

1 2 3 4 5

Week 0 2 4 6 8 10 12

Protein immunization

Animal Study Schedule – Intact and Mixed Envelope

Protein Vaccination

Protein vaccination:

Dose: 75µg of protein formulated with 90 units of ISCONMATRIX

Route: intramuscular injection

Serum sample collections

T e m p o ra l a g a in s t in ta c t g p 1 2 0 Im m u n o g e n R e s p o n s e -2

W e e k s

gp

12

0-s

pe

cif

ic I

gG

tit

ers

0 2 4 6 8 1 0 1 2

1 0 3

1 0 4

1 0 5

1 0 6

1 0 7

g p 1 2 0 -B In ta c t

g p 1 2 0 -B M ix e d

V a c c in e g ro u p s

A.

T e m p o r a l a g a in s t M ix e d g p 1 2 0 Im m u n o g e n R e s p o n s e -2

W e e k s

gp

12

0-s

pe

cif

ic I

gG

tit

ers

0 2 4 6 8 1 0 1 2

1 0 3

1 0 4

1 0 5

1 0 6

1 0 7

g p 1 2 0 -B In ta c t

g p 1 2 0 -B M ix e d

V a c c in e g ro u p s

B.

Temporal antibody responses

against gp120-B intact

against gp120-B mixed

gp

12

0-s

pe

cif

ic I

gG

tit

er

1 0 3

1 0 4

1 0 5

1 0 6

1 0 7 E n v-A ( in ta c t) E n v -A (m ix e d )

R a b b it g ro u p s

A -in ta c t A -m ix e d A -in ta c t A -m ix e d

g p 1 2 0 c o a tin g a n tig e n

gp

12

0-s

pe

cif

ic I

gG

tit

er

1 0 3

1 0 4

1 0 5

1 0 6

1 0 7 E n v-B ( in ta c t) E n v -B (m ix e d )


B -in ta c t B -m ix e d B -m ix e d


B -in ta c t

gp

12

0-s

pe

cif

ic I

gG

tit

er

1 0 3

1 0 4

1 0 5

1 0 6

1 0 7 E n v-C ( in ta c t) E n v -C (m ix e d )


C -in ta c t C -m ix e d C -m ix e d


C -in ta c t

gp

12

0-s

pe

cif

ic I

gG

tit

er

1 0 3

1 0 4

1 0 5

1 0 6

1 0 7 E n v-E ( in ta c t) E n v -E (m ix e d )


E -in ta c t E -m ix e d E -m ix e d


E -in ta c t

Intact Mixed gp120-B vaccine

Im m u n o -re s p o n s e a g a in s t fu l l- le n g th V 3 -J R F L

V3

-sp

ec

ific

Ig

G t

ite

rs

1 0 3

1 0 4

1 0 5

1 0 6

in ta c t im m u n o g e n (g p 1 2 0 -J R F L )

m ix te d im m u n o g e n (g p 1 2 0 -J R F L )

V3-specific antibody responses

against V3-B

NAb titers against SF162

In ta c t M ix e d

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

g p 1 2 0 -B

V a c c in e g r o u p s

NA

b t

ite

rs

Combined all 4 intact immunogen groups and 4 mixed immunogen groups

In ta c t M ix e d

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

g p 1 2 0 -4 im m u n o g e n s


NA

b t

ite

rs

Combined all 4 intact immunogen groups and 4 mixed immunogen groups

In ta c t M ix e d

0

1 0

2 0

3 0

g p 1 2 0 -4 im m u n o g e n s b re a d th


% v

iru

se

s n

eu

tra

liz

ed

HIV-1 pseudoviruses Clade B - Sensitive: SF162 Clade A: Q23 ENV17 Clade B: SS1196, 6535, QH0692 Clade C: ZM109, ZM197

0.E+00

5.E+04

1.E+05

2.E+05

2.E+05

3.E+05

1

21 41 61 81

10

1

12

1

14

1

16

1

18

1

20

1

22

1

23

7

25

7

27

7

29

7

31

7

33

7

35

7

37

7

39

7

41

7

43

7

45

7

47

9

M

E

C

B

A

JPT peptide microarray

C1 V1 V2 C2 V3 C3 V4 C4 V5 C5

0.E+00

5.E+04

1.E+05

2.E+05

2.E+05

3.E+05

1

21 41 61 81

10

1

12

1

14

1

16

1

18

1

20

1

22

1

23

7

25

7

27

7

29

7

31

7

33

7

35

7

37

7

39

7

41

7

43

7

45

7

47

9

M

E

C

B

A

Env-B Intact

Env-B Mixed

Conclusion for Strategy One

- Bad news

- Very hard to control clipping during cell culture

- Good news

- Clone B gp120 protein is the only one with V3

clipping

- No significant difference in both antigenicity and

immunogenicity between mixed gp120 vs. intact

gp120

Strategy Two

- downstream removal of proteases and

reduction of gp120 clipping

Optimization with Purification Activities

- AEX optimization - pH/NaCl – identified optimal pH

- EDTA to increase stability of gp120

- Low pH additives to increase stability

- CEX optimization - Buffers (three tested) to reduce low MW

contaminates

- Urea (four concentrations) to reduce degradation

- Gradient vs. step elution

- Additives (arginine)

Purification Activities (cont.) Alternative columns

- Capto MMC (GE)

binding/elution screen, gradient elution, wash conditions - Mx-Trp

Binding/elution screen, gradient elution - POROS 50 HS

- Benzamidine affinity resin

Initial tests, optimization, characterization of FT - HIC

Post AEX material, step/gradient, - Phenyl borate affinity resin

Initial tests Conclusion

- None of the above activities was effective in significantly reducing clipping

A “novel” treatment strategy

Cation precipitation removed contaminating enzymes Western blotting 12/2013 with Anti-Fucosidase antibody

Cation 50 mM

Super

Cation 50 mM

Pellet

Cation 175 mM

Pellet

Cation 150 mM

Pellet

Cation 100

mM Pellet

Cation 175 mM

Super

Cation 100 mM

Super

Cation 150

mM Super

Marker Starting Material

Cation 50 mM

Super Reduced

Cation 50 mM

Pellet Reduced

gp120B-3LSUB1

50L SUB

Clarification (Depth Filtration)

UF/DF (TFF1)

Anion Exchange Chromatography

gp120 in FT

Cation Exchange Chromatography pH 4, gp120 Bind/Elute

UF/DF (TFF3)

Size Exclusion Chromatography

Revised gp120 Purification Process Flow Diagram (Dec 2013)

Viral Filtration

= viral clearance step

UF/DF (TFF2) UF/DF (TFF4)

Fill Cationic Precipitation Depth Filtration

220

160

120 100 90 80 70 60

50

40

30

25 20

15

10

(kD)

Comparison of Old and New (Cationic Precipitation) Purification SEC Fractions

Dimer

Monomer

Gp120 or

Contaminating

Proteins?

220

160

120 100 90 80 70 60

50

40

30

25 20

15

10

(kD)

• NON-REDUCED SDS-PAGE • Co-eluting contaminants gone in new process

Old Purification Method New Method w/ Cationic Precipitation

Final tox lot gp120A3 BDS gp120A Reference Standard

gp120A3 BDS

NR R NR R

220

160

120

90

80

70

60

50

40

30

25

20

15

10

(kD)

100

• 1ug/lane

• 4-12% Bis-Tris Gels

• MES Buffer

• Simply Blue Stain

Conclusion for Strategy Two

Further process development work was able

to identify the cation precipitation method to

greatly remove impurities and improve the

quality of final purified gp120 proteins

Final gp120 proteins are >99% purity and

85% monomer for all four clones

• Director

– Derek Hei

• Manager of Manufacturing

– John Welp

• Protein Purification Team

– Chris Bartley (Project Manager)

– Lisa Burdette

– Bryan Atkinson

– Nicole Vike

– Sonia Petty

Acknowledgements Waisman Biomanufacturing

• Manager of Quality Assurance

– Tim Sparks

• Quality Assurance Team

– Jennifer Jauquet

– Jaime Bellon

• Quality Control Team

– Ross Meyers (Team Lead)

– Janice Boyer

– Paul Brisco

– Joshua Sotos

– Megan Fitzgerald

– Devin Green

• Business

• Kari Thostenson

U Mass Med School

Shixia Wang

Dong Han

Aaron Wallace

Shengqin Wan

Guangnan Hu

Abigail Miller

Cindi Callaghan

Jill Serrano

Gary Ostroff

Consultants

Debra Barngrover

Eric Wiechert

Indresh Srivastava

Sean Du

Funding Support

NIH/NIAID/DAIDS

IPCAVD

Michael Pensiero

Vijay Mehra

Vijaya Rangavajhula

Jane Halpern

Tina Tong

Joellyn Bowser

CAVD/BMGF

Amy Weiner

Acknowledgements SAB Members

Carl Alving

Michael Keefer

Wayne Koff

Richard Kornbluth

Stanley Plotkin

John Shiver

Edward Arcuri

Phillip Berman

Michael Brennan

Bharat Dixit

Shiu-Lok Hu

HIV-ePro

HIV Envelope Protein Production Program Waisman Biomanufacturing (WB) and the University of Massachusetts Medical

School (UMMS) have partnered to form HIV Env Protein Production Program

(HIV-ePro).

Our goal is to provide essential manufacturing and development consultation

services to the global HIV vaccine field to address the unique challenges

associated with cGMP production of HIV-1 Env proteins including structure

complexity, sequence diversity and lack of well-established methods for producing

and characterizing Env proteins for use in vaccine clinical trials.

20+ years’ experience in HIV-1 Env research and vaccine development; led previous program to produce world’s 1st polyvalent Env protein formulation under GMP conditions

Most updated technology platform to produce high yield, high purity and high quality recombinant HIV-1 Env protein vaccines in mammalian expression system

Available MCB and GMP grade recombinant gp120 proteins from major subtypes Consultation on cell line development, GMP manufacturing, potency assays, QC and

stability analysis of recombinant Env proteins for clinical studies Ability to develop customized processes that are reproducible and fully scalable, yielding

end products with support of unique reagents and assays Comprehensive Chemistry, Manufacturing, and Control (CMC) support for Investigational

New Drug (IND) Filings

gmp production of a polyvalent recombinant gp120 … · elisa, western blotting rt -pcr + pcr/ qpcr...

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