Biologicals manufacturing summit_feb_2005 _ a case study in strain and process development

Failure and Success in the Clinic- a case study in strain and

process development

Biologics Manufacturing Summit24 February 2004

Overview

• Introduction to recombinant albumin• Phases of recombinant albumin

development• Clinical trials data• Back to the drawing board• More clinical trials

Recombinant Human Albumin

• Large secreted protein

– 67kDa– 585 amino acids

• Highly folded– 35 cysteines– 17 disulphide bonds– 1 free cysteine

Structure of rHA with five molecules of myristate bound.

Curry et al. (1998) Nature Structural Biology 5, 827-835

Recombinant Human Albumin

• Produced using yeast, Saccharomyces cerevisiae

• 3,500m2 cGMP compliant facility dedicated to the manufacture of rHA

– 8000L fermenter, chromatography columns up to 100L, 25 process vessels of up to 13000L

• Free of animal-derived products• FDA audited

Yeast – Positive Attributes

• GRAS status– S. cerevisiae– K. lactis

• Wide range of strains• Extensive industrial history

– 16 S. cerevisiae therapeutic products marketed

– 7 P. pastoris therapeutic products under development

Gerngross, T. (2004) Nature Biotechnology 22, 1409-1414

8m3 working volume fermentation vessel

Nottingham, U.K.

Mitotically Stable Vector Systems

• Whole 2µ plasmids– Disintegration vector + rHA

pDB2244, cirO

0

1

2

3

4

5

DB1

DS65

DS212

DS569

DS1101 D88

DXY1

D540

D638

D674

rHA

pro

duct

ivity

g/L

yap3- hsp150- pmt1-

rHA producing yeast strains obtained byaspecific mutagenesis

1,2,7,8-diepoxyoctane (DEO)N-methyl-N'-nitro-N-nitrosoguanidine (NTG)4-nitroquinoline N-oxide (NQO)

Strains obtained by acombination of specific &aspecific mutagenesis

DEONTG

NQO

NTG

NTG

Yeast Strain Family

*

* Productivity of monomeric albumin assessedby densitometry / SDS PAGE

Process Variations

Dates of Manufacture 1992-1994 1994-1997 1999 to dateOrganism D88 D540 D638Harvest Centrifugation Centrifugation Centrifugation

Cation exchange Cation exchange Cation exchange

Affinity Anion exchange Anion exchange

UF Affinity Affinity

GPC UF UF

Anion exchange GPC Affinity

Affinity UFCation exchange 2

Anion exchange 2

FUFFormulation

Product rHA-stage1 rHA-stage2 Recombumin(R) 20%

Downstream Process

Clinical Studies

rHA-Stage 1 Clinicals

• Healthy male volunteers– Successfully administered up to 50g i.v

• Yeast sensitive volunteers– Skin prick positive to yeast– 17 of 18 patients successfully dosed– Final patient suffered an anaphylaxis

• Consequently redevelop the product from beginning to end

rHA-Stage 2 Development

• Improve safety and purity– Reduce yeast antigen levels

• Improve process and economics– New yeast strain with increased rHA

expression level– Improved downstream process

• New chromatography steps• Improved buffer washes• Ensure process integration

Downstream Process Improvement through Expression Strain Modifications

YAP3

yap3

rHA monomer

45kDa fragment

-Phe-Gln-Asn-Ala-Leu-Leu-Val-Arg-Tyr-Thr-Lys-Lys-Val-Pro

•45kDa N-terminal fragment

•Observed in Pichia sp,

Kluyveromyces sp and Hansenula sp

•Carboxy terminus heterogeneous

•Terminating between Phe403 and Val409;

most common Leu407 and Val409

0

1

2

3

4

5

DB1

DS65

DS212

DS569

DS1101 D88

DXY1

D540

D638

D674

rHA

pro

duct

ivity

g/L

yap3- hsp150- pmt1-

rHA producing yeast strains obtained byaspecific mutagenesis

1,2,7,8-diepoxyoctane (DEO)N-methyl-N'-nitro-N-nitrosoguanidine (NTG)4-nitroquinoline N-oxide (NQO)

Strains obtained by acombination of specific &aspecific mutagenesis

DEONTG

NQO

NTG

NTG

Yeast Strain Family

*

* Productivity of monomeric albumin assessedby densitometry / SDS PAGE

Downstream Process Improvement through Expression Strain Modifications

• Hsp150p (Pir2p)– Host cell wall protein– Large

• ~150kDa • extensively O-linked

glycosylated• 47kDa deglycosylated

– Removed by gel permeation chromatography

– Antigenic in yeast sensitive subjects

Enrichment by ConAchromatography

HSP150+ HSP150-

0.2m

g

2mg

10m

g

0.2m

g

2mg

10m

g

Western blot with anti-Hsp150p

Russo et al. (1992) PNAS 89, 3671-3675

Process Variations

Dates of Manufacture 1992-1994 1994-1997 1999 to dateOrganism D88 D540 D638Harvest Centrifugation Centrifugation Centrifugation




GPC UF UF



Anion exchange 2

FUFFormulation


Downstream Process

rHA-stage 2 Studies (1995-6)Intravenous

Study Number 001 002

Subjects 36 healthy males

6 healthy males

Dose ascending dose 1.5, 3, 6, 12.5, 25, 50 g 4 x 25 g

Route of Administration

single i.v. Aug - Sep 95

multiple i.v. Oct 95 - Jan 96

rHA-stage 2 Studies (1997) i.m and s.c

Study Number CE 1143/1-1001 CE 1143/1-1002

Subjects * 40 rHA 20 HSA

40 rHA 20 HSA

Dose 200 mg (0.8 mL)

200 mg (0.8 mL)

Route of Administration s.c. i.m.

Study Days 1, 8, 15, 22, 29, 57 1, 8, 15, 22, 29, 57

Follow-up 1 month 1 month

* healthy male and female volunteers

rHA-stage 2 Studies (Phase I: 1997)

• 134 subjects dosed:– s.c. study: total 64 (43 rHA, 21 HSA)– i.m. study: total 70 (47 rHA, 23 HSA)

• 7 non-serious allergic reactions:– s.c. study: 3 rHA (7.0%)– i.m. study: 3 rHA (6.4%), 1 HSA (4.3%)

• Overall incidence of reactions unexpected – between study day 23-32 after 4-5 repeated

doses– with a delay of 3 days after the last dosing

• Delayed type hypersensitivity to rHA-stage 2

i.v., i.m and s.c Clinical Summary

• rHA-stage 2 well-tolerated i.v. at doses up to 50g or 4 x 25g, but excess of allergic reactions following repeat administration s.c. or i.m.

• Product purity requirements appear to be significantly higher for repeat administration s.c. or i.m. than for i.v. infusion

• Research program of product improvement studies to reduce yeast-derived impurities

– Research program resulted in Recombumin®

20%

Targets for Product Improvement

• Yeast antigens– improved purification process

• Very low level of mannosylated rHA– improved host strain– improved purification process

• Nickel ions– improved purification process

• rHA-ADH fusion protein (theoretical)– improved expression plasmid

Conclusion of Clinical and In Vitro Investigations

• Intradermal test, T-cell proliferation and serology indicated involvement of yeast antigens

• Serology indicated possible involvement of mannosylated rHA

• No indication of involvement of nickel ions or fusion protein in allergic reactions

Translational read-through

L G L stop A L D F F A R G 34aa S K stopTTA GGC TTA TAA GCT TTG GAC TTC TTC GCC AGA GGT...........TCT AAA TAA ..

C-Terminus Albumin ADH1 Terminator

L G L stop stop A stopTTA GGC TTA TAA TAA GCT TAA TCC ..........

C-Terminus Albumin ADH1 Terminator

Anti-Adh1p immunoaffinity purificationrHA-Adh1p rHA

Load

Fl T

hru

Elua

te

Load

F Th

ru

Elua

te

• Estimated translational read-through– 0.002% (w/w) rHA-Adh1p fusion

Changes to Expression Plasmid

• Demonstrated that rHA-ADH fusion protein present at approx. 0.002% w/w rHA

– due to read-through of translation stop codon

• New rHA expression plasmid constructed– Double stop codon (UAA UAA) replaces single stop

codon (UAA)– 132bp of ADH1 coding region at the 5’ end of the

ADH1 terminator has been removed

• Sequence of entire expression cassette verified

Mannosylated rHA

• Approx. 0.7% of rHA binds to Con A– Due to O-glycosylation with mannose– Average of 3-5 moles/mole– Linkages α-1,2 and α-1,3. No evidence

of branching– Twelve potential sites of modification

identified

Mannosylated rHA cont.

• Reduction in m-rHA– New yeast strain, pmt1– Improved downstream process

• pmt1 mutant– Shorter glycoforms

• Additional chromatography steps– Reduced the amount of Con A binding

material five fold

Effect of pmt1 mutation

Amount of conA-binding rHA reduced approx. five-fold

conA eluate from wild type-derived rHA

conA eluate from pmt1 mutant-derived rHA

66500 67000 67500 68000mass0

100

%

66500 67000 67500 68000mass0

100

%

UnmodifiedrHA

+1

+2

+3

+4

+5

+6

+7

+8

Further Quality and Purity Improvement

• Reduction in yeast antigens– Additional chomatography steps– Improved buffer washes

• Reduction in trace Nickel ions– Change to FUF conditions

Improved Product - yeast antigens

1 2 1 2

• High sensitivity Western blot

• Load: 50µg rHA

• A : antibodies raised to new strain

• B: antibodies raised to former strain

• 1. Former product

• 2. Improved productA B

Process Variations

Process Method PD04 PD09 PD12Dates of Manufacture 1992-1994 1994-1997 1999 to date

Organism D88 D540 D638Harvest Centrifugation Centrifugation Centrifugation




GPC UF UF



Anion exchange 2

FUFFormulation


Downstream Process

Improvements in Product Quality

• Yeast antigens– Reduced approx. 10-fold by ELISA– Significant improvement in Western blot

• Mannosylated rHA– Reduced approx. 5-fold in final product– Reactivity with AE subjects’ antibodies reduced

by a factor of between 4 to >20– Combined reduction in reactivity of Recombumin

>20-fold• Nickel ions

– Reduced to HSA levels

Recombumin® 20% rHA Clinical Studies

• Phase I clinical study to verify safety for use as an excipient via i.m. administration

– 5 x 65 mg repeat doses evaluated in 300 subjects (150 rHA, 150 HSA)

– Comparison of tolerance of Recombumin® 20% and HSA

• Phase I study to demonstrate safety and tolerance i.v.

Recombumin® 20% rHA Phase I Study Overview (1999-2000)

5 mg50 rHA

15 mg

65 mg 50 HSA

50 HSA

Highesttolerated dose

100 rHA100 HSA

50 HSA

50 rHA

50 rHA

65 mg

5 mg50 rHA

15 mg

65 mg 50 HSA

50 HSA

Highesttolerated dose

100 rHA100 HSA

50 HSA

50 rHA

50 rHA

65 mg65 mg

Prospective, randomized, double-blind, active controlled (HSA), Repeated (5 times weekly intervals) i.m. administrations, 3 dose levels, performed under US IND

Phase I Intramuscular Study Summary

• Product improvement to reduce yeast-derived impurities was successful

• Demonstrated comparable safety and tolerability of Recombumin® 20% rHA and HSA via i.m. administration*

*Bose et al (2005) J Clin Pharmacol 45:57-67

Phase I Intravenous Study (2001)

15 rHA50 g

Prospective, randomized, double-blind, sex-stratified, HSA controlled,dose-escalating, repeated i.v. administrations, 3 dose levels

15 HSA

20 g10 g

Phase I Intravenous Study Summary

• Dosing and follow-up completed successfully

• No serious or critical adverse events observed

• No measurable IgG or IgE antibody responses

Comparison of Yeast Impurities inRecombinant Yeast-Derived Products

Product Dose GM-CSF 4 ng

Hep. B Vaccine 10,000 ng

Hep. B Vaccine(2) 100 ng

1.6 ng (65mg clinical dose)

Recombumin® 20% 0.005 ng

(Vaccine 0.2 mg rHA)

Non therapeutic uses of albumin

•Final product excipient–Recombinant proteins–Vaccines

•Cell culture•Diagnostic imaging

–Microspheres - ultrasound–Radiolabelled

•Coating of medical devices•In vitro fertilisation media

• “Recombinant albumin was equivalent to native serum albumin in its capacity to protect immunological, biological and biochemical properties of TSH, IL-15 and G-CSF”

• “The recombinant form of albumin … should be considered as a suitable stabilizer in the preparation of lyophilized products and reference reagents”

Biologicals (1998) 26, 331-346

FDA Statement

• At a recent FDA/PDA meeting, a presentation was given entitled “Bovine-derived Products Used in the Manufacture and Formulation of Vaccines: current Policies and Issues for the Future”. In that presentation by W.M. Egan, Acting Director, Office of Vaccines, FDA, an endorsement of the use of recombinant albumin in place of HSA was made under Areas to Consider;

• Consider “Replacement of human serum albumin (HSA) with recombinant albumin”.

Summary

• Recombumin® 20% rHA is a consistent, high purity product.

• Recombumin® 20% rHA is structurally identical to HSA.

• Recombumin® 20% rHA provides a safe alternative to plasma-derived albumin or to gelatin for use in the manufacture of pharmaceutical products.

Dr S Berezenko

[email protected]

Biologicals manufacturing summit_feb_2005 _ a case study in strain and process development

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