EVI rendez-vousNov. 14th, 2011

Stephan Hellwig

Process development &GMP production of

pfAma1 DiCo1-3 clinical-grade API

Efficient project communication among 3-5 partners fromdifferent countries was achieved by ca. 60-80 Teleconferences and only ca. 5 face-to-face meetings

Cell separation and FiltrationFollowing High-cell-density Pichia

fermentation

Downstream processing of the API in class C cleanrooms

Release testing of the API or purified bulk

What‘s behind the phone ?1 batch of each pfAma1 DiCo API was

produced in accordance with GMP andfilled in aliquots for further processing.

Project progress12-2008: First contact

01-2009: Quotation WP 1 (Non-GMP PD)

06-2009: Contract signed

07-2009: First fermentation

02-2010: Audit NNE Pharmaplan

04-2010: Manuf. & qualif. of MCBPreparation of Manuf. Instruction

06-2010: Finalization of WP 1 (Non-GMP)

06-2010: First technical run in cleanroomFinalizing Manuf. Instruction

08-2010: GMP contract signed

11-2010: GMP campaign (4 weeks)

Until now: Release QCFormulation developmentDP manufacturing

Process developmentProcess development

The infamous degradations

-0,4

-0,2

0

0,2

0,4

0,6

0,8

1

1,2

1,4

0,0000001 0,000001 0,00001 0,0001 0,001 0,01

E40

5

1/Dilution

Supernatants PPF 122, est. ca. 90 mg/L in harvest supernatant

SN122-4

SN122-5

SN122-8

SN122-7

SN-122-8

SN122-9

SN122-10

SN122-11

STandard

Early & Final fermentation protocols –trading off yield for quality

0 12 24 36 48 600

20

40

60

80

1000

500

1000

1500

Dis

solv

ed o

xyge

n [%

]Te

mpe

ratu

re [°

C]

Time [h]

StartMeOH feed

StartGY feed

50%

w/v

Gly

cero

l Fee

d +

Ptm

1 [m

L]M

eOH

feed

+ P

tm1

[mL]

12.5

% N

H4O

H [m

L]

0

50

100

150

200

250

300

350

baseFW

OD

600,

FW [g

·L-1]OD

5,50

5,75

6,00

6,25

6,50

PPF 109 AMVAC_01_ND1_20 L

pH

0 12 24 36 48 600

50

100

150

2000

150

300

450

600

750

900

Dis

solv

ed o

xyge

n [%

]Te

mpe

ratu

re [°

C]

Time [h]

StartMeOH feed

StartGY feed

50%

w/v

Gly

cero

l Fee

d +

Ptm

1 [m

L]M

eOH

Fee

d +

Ptm

1 [m

L]25

% N

H4O

H [m

L]

0

50

100

150

200

250O

D60

0

FW

[g·L

-1]Base

FW

OD

5,50

5,75

6,00

6,25

6,50

PPF 134 AMVAC_01_ND3_20 L

pH

Media comparison

Component Invitrogen 1st gen. IMEPhosphoric acid (85 %)

23.7 mL 23.87 mL 4.25 mL

CaSO4∙2H2O 1.18 g 1.05 g 0.18 gK2SO4 18.2 g 18.28 g 2,81 g MgSO4 7H2O 14.9 g 14.96 g 2.31gKOH 4.13 g 4.15 g 0.72Glycerol (1.26 g/mL) 40 g 40.17 g 50 mLEDTA 0 g 0.150 g 0.150 gPtm1 Trace salts 4.35 mL 4.76 mL 4 mL

Component Invitrogen 1st. gen (Low Cu) IME

CuSO4∙5H2O 6.0 g 0.6 g 0.6 gNaI 0.08 g 0.08 g 0.08 gMnSO4∙H2O 3.0 g 3.0 g 3.0 gNa2MoO4∙2H2O 0.2 g 0.2g  0.2 gBoric acid 0.02 g 0.02 g  0.02 gCoCl2∙6H2O 0.5 g 0.92 g 0.2 gZnCl2 20 g 20 g 20 gFeSO4∙7H2O 65 g 65 g 65 gBiotin 0.2 g 0.2 g 0.2 gH2SO4 conc. 5 mL 5 mL 5 mL

Stability after IMAC 1 – determining hold steps

N-terminal alternatives

Gradient‐Elution HIC

Coloration in Ama1 DiCo DSP

1st generation vaccine process history report describes that :• Butyl-eluate still contained Pichia pigments but in a less extent than IMAC-eluate. Butylchromatography was thus kept as second purification step.

IME made the very same observation, but recommendation from QA and QP is to investigate the nature of the contaminant or to remove it

Coloration in Ama1 DiCo DSP

Lots of experiments to get rid of colouration Is the protein brownish ?

Early (non-GMP) purified bulk stability

Looking into the future at an early point in development: stability of formulated

lyophilized purified bulk

Scaleup & Transfer to GMP

Scaleup & Adaption to GMP

Total processing time: ca. 6‐8 hoursTotal work day: ca. 8‐12 hours

Temperature issues duringprocessing ?

Disposables cost approx. 850 €bags, ca. 800 € filters

Possible layout of cell separation

CARR

product

filtrate1,5 µm

Filter1,5 µm

bulkharvest

Filter0,22 µm

Carrcentrate

08.09.2010, ca. 24 h, ca. 2/3 of bulk harvest processed

Post manufacturing of drug substance

Release testing issues

• DiCo‐specific assay to quantify each API in the mix:• DiCo‐specific ELISA• RP‐HPLC• BiaCore

• Host‐cell proteins• Commercially available „Cygnus“‐Kit not useful (cross‐reactivity)

• Dimers & Aggregates

Drug substance formulation development: Analysis of aggregates and dimers

Drug substance to drug product: Formulation & Lyophilization

• Aggregates occurduringlyophilization

• Dimers occur, time‐dependantand possiblyoxygen‐dependant in PP vials, but not in EVA bags.