pasteurisation inactivates coagulation enzymes … inactivates coagulation enzymes during flebogamma...

1

Plasma Product Biotechnology Meeting, May 19-13, 2011

Pasteurisation Inactivates Coagulation Enzymes

During Flebogamma® and Flebogamma® DIF

Production

Marta José1, Nuria Marzo1, Mariona Bono2, Marta Carretero3, Maite López1,

Pere Ristol1and Juan I. Jorquera1

1Research and Development, Instituto Grifols and 2Diagnostic Grifols; 3Clinical Trials and

Pharmacovigilance, Instituto Grifols. Parets del Vallès, Barcelona, Spain

2

• The starting material for current IVIG production can

be Cohn’s fraction II+III or fraction I+II+III (or the

“equivalent” intermediates from Kistler-Nitschmann

fractionation).

Starting Material: Cohn, Kistler-Nitschmann

3

17% Ethanol, pH 5.2

Fr-II+III Precipitate

A+Fr-I

Fr-II+III-W

Fr-III (or I+III) Sup

Fr-II

Precipitate B Sup

Precipitate GG

Fr-II+III Sup

(0.3-0.4 g/L IgG)

Ppt. A Sup

(~0.1 g/L IgG)

W

Fr-III (or I+III) Sup (PTC and other impurities)

Precipitate B Sup (PTC and other impurities)

12% Ethanol, pH 5.1

Yield = ~ 60% Yield = ~ 80%

(by-pass)

(by-pass: 75%)

COHN – ONCLEY (method 9, modified) KISTLER - NITSCHMANN

Cryoprecipitate Supernatant

Cohn-Oncley vs. Kistler-Nitschmann

(basic methods to produce Intramuscular IGs)

Fr-I

Intramuscular IGs gave rise to severe adverse events when infused intravenously:

Fraction II or precipitate GG need further processing to obtaing IVIG

Example of immunoglobulin fractionation processes

4

In addition to complement

components, Fraction I

contains important amounts

of blood coagulation

factors.

Both groups of proteins

include proenzymes that,

when activated, participate

in inflammation and blood

clotting processes.

Curling J (Ed.) Methods of Plasma Protein Fractionation. London: Academic Press, 1980

Starting Material

5

Coagulation cascade: feedback activation

KAL PK

HK HK

XII XIIa

XI XIa Ca2 +

II Ca2 +

IIa

Fibrinogen Fibrin

Tissue factor

VII VIIa Ca2 +

IX IXa

X Xa

Under physiological

conditions, some

steps also require

phospholipids and/or

other cofactors

6 Amara U, et al. J Immunol 185:5628,2010

Coagulation & Complement

Coagulation and Complement serine proteases have the

capacity to interact and activate each other

7

1962

1983.

IGIV preparation strategies: e.g. Acid pH treatment

8

A typical procedure uses Anion

Exchange for IVIG production but

this step may fail to remove some

unwanted impurities, depending

on the starting material and the

specific purification conditions

Effluent (IgG and residual impurities with positive charge)

IgA (-)

Alb (-)

IgM (-)

Trf (-)

(others)

Flow through

IgG (+); FXI/FXIa (+); FIIa(+); Others (+; FVII?; FXII?; Kallikrein?; )…

PKA (-)

FII (-)

FIX (-)

FX (-)

Resins with positive charge

Impurities with negative charge

Starting Material

9

Isoelectric points of plasma

proteins or enzymes

Protein/Enzym Ip Reference

IgG 6.3-7.3 Hill et al. Principles of Biochemistry 7th edition. Mc GrawHill, 1983

Thrombin 6.4-7.6 Fenton, et al. JBC (1977) 252:3587-98

FII

(Prothrombin) 5.64 Swiss Institute of Bioinformatics. ExPASY Proteomics Server (www.expasy.org)*

FVII 6.9 Swiss Institute of Bioinformatics. ExPASY Proteomics Server (www.expasy.org)*

FIX 5.3 Swiss Institute of Bioinformatics. ExPASY Proteomics Server (www.expasy.org)*

FX 5.7 Swiss Institute of Bioinformatics. ExPASY Proteomics Server (www.expasy.org)*

FXI 9.1 Heck et al. J Exp Med 1974 140:1615-30

FXII 8.0 Swiss Institute of Bioinformatics. ExPASY Proteomics Server (www.expasy.org)*

PKA 4.2-4.4 Alving et al. N. Engl. J. Med. (1978) 299: 66-70

Kallikrein 8.6 Swiss Institute of Bioinformatics. ExPASY Proteomics Server (www.expasy.org)*

Tissue Factor 6.6 Swiss Institute of Bioinformatics. ExPASY Proteomics Server (www.expasy.org)*

*: Predicted from primary sequence

10

• The thromboembolic adverse events (TEEs) have been linked to several different potential causes.

• However, there was no solid demonstration of any specific isolated cause for these events.

• The recent TEEs outbreak is probably the incident where a clearer relationship to a single cause (presence of procoagulant factors – including FXIa-) is being established, but additional impurities may also be involved.

IVIGs and TEEs

11

Production Processes

12

Plasma

Cryoprecipitate

Cryosupernatant

Fraction I (Discarded)

Fraction I Supernatant

Fraction II+III (IVIG)

Fraction IV1+IV4

Fraction IV1+IV4 Supernatant

Fraction V

Fraction II+III Supernatant

Grifols’ basic (Cohn) fractionation

IVIG starting material: Fr II+III

13

Specific VI step

FLEBOGAMMA® DIF

(double PEG purification)

PEG+IEX Column Chromatography

Acid pH

20 nm Planova NANOFILTRATION

PEG Fractionation (TFF)

SD-treatment

Purification & Non-specific VI step

PASTEURIZATION

Purification & VI step

Pasteurization requires removal

of aggregates (using PEG

precipitation)

leading to very high purity

products

Grifols IVIG Production Process

15

PASTEURIZATION INACTIVATES SERINE PROTEASES

(Clotting proenzymes and Complement components are serine proteases)

High serine protease activity

Purification process

16

Analysis of the coagulant activity

–in vitro methods-

• Activated factors (NaPTT, Ph.Eur.), “generic” (PPP) and with FXI deficient plasma.

• Thrombin presence (Ph.Eur.).

• Determination of prekallikrein activator (PKA) and kallikrein (or “kallikrein-like” serine proteases), Ph.Eur.

• Thrombin Generation Test (TGT, Technoclone), “generic” (PPP) and with FXI deficient plasma.

• Enzyme Immunoassay for FXI Antigen (FXI:Ag, Dunn Labortechnik).

• Coagulation (pro)enzymes (one stage APTT or PT assay):

– FII, FVII, FIX, FX, FXI and FXII.

17

Coagulation factors in Fraction II+III (more frequent starting material for IVIG production)

% FII FVII FIX FX FXI FXII

Plasma pool 100 100 100 100 100 100

Fraction II+III

suspension

LOT A 68 201* 67 33 92 40

Fraction II+III

suspension

LOT B 61 121* 70 15 76 35

Total % recovery of (pro)enzymes in industrially resuspended Fraction II+III

versus the plasma pool (assuming that plasma contains 1 U/ml for all factors).

FVII recovery (>100%) may be due to the presence of activated FVII.

The presence of other activated enzymes cannot be discarded.

18 Fr II+III industrial extraction sample.

NaPTT PPP

(n=4)

Thrombin presence

(n=3)

PKA

(n=4)

“Kallikrein”

(n=3)

TGT PPP (n=1)

Vehicle:

421 nM IIa

TGT FXI-Def. (n=1)

Vehicle:

0 - 17 nM IIa

sample/control

Ratio

1/5 dilution assay

6h 37ºC 24h 25ºC IU/ml ∆Abs/min nM Thrombin nM Thrombin

0,12 - 0,86 1/3 lots

positive

1/3 lots

positive 137-3977 0.14 - 1.4 620 425

Coagulation activation status in Fraction II+III

All tests indicate that FrII+III contains activated coagulation

factors, like thrombin, PKA, “Kallikrein” and possibly FXIa.

20

Clotting (Pro)enzymes content evolution in

Flebogamma® DIF process (n=2-3)

FII FVII FIX FX FXI FXII

Fraction II+III

suspension 57-65 113-192 64-65 14-31 71-88 33-38

4% PEG Filtrate not

quantified

not

quantified

not

quantified

not

quantified

not

quantified

not

detected

DEAE effluent not

quantified

not

quantified

not

quantified

not

quantified

not

quantified

not

quantified

Concentrated - UFI not

quantified

not

quantified

not

quantified

not

quantified 0.4 to 1.6

not

quantified

After

Acid treatment

not

quantified

not

quantified

not

quantified

not

quantified

Not

quantified

to 0.8

not

quantified

After

Pasteurisation

not

quantified

not

quantified

not

quantified

not

quantified

not

quantified

not

quantified

FINAL BULK

(5 and 10%)

not

quantified

not

quantified

not

quantified

not

quantified

not

quantified

not

quantified

(IU per gram of total protein)

• Fr II+III suspension has variable amounts of all coagulation (pro)enzymes.

• Downstream, only FXI(a) is detected occasionally at low levels in concentrated materials.

• Pasteurization inactivates the residual FXI(a).

21

Flebogamma® DIF purification process follow-up

Resusp.

FrII+III (n=4)

PEG 4 %

filtrate (n=3)

DEAE

effluent (n=3)

Concentr

ated UFI

(n=3)

After acid

treatment

(n=3)

After

pasteu.

(n=3)

Concen.

final bulk

(n=3)*

FXI:C (IU/g protein) 71-88 <0.007 <0.007 0.4-1.1 <0.007-0.8 <0.007 <0.007

NaPTT-PPP (s) 17-135 219-241 218-288 197-213 220-250 225-336 225-299

PKA (IU/ml) 137-3977 34-143 <2 <2 <2 <2 <2

"Kallikrein"

(AU/min) 0.14-1.40 0.001-0.004 0.003-0.004

0.017-

0.030 0.013-0.024

0.001-

0.002 0.000-0.002

TGT (nM)

PPP 199 12 -8 56 19 Not

detected

Not

detected

FXI-Def (n=3) 474±57 53±15 41±14 141±14 135±39 Not

detected

Not

detected

*5% bulk (n=1) plus 10% bulk (n=2). NaPTT control: 237 to 301 seconds. TGT-PPP n = 1.

22

Flebogamma DIF process follow-up:

TGT FXI deficient plasma

UFI

UFIII 10 % bulk

After

pasteurization Vehicle

After acid

treatment

Fraction II+III

suspension

0

100

200

300

400

500

Pe

ak

of

thro

mb

in a

cti

vit

y (

nM

)

23

Analysis of the procoagulant activity in

Flebogamma® DIF -Summary-

• The suspension of Fr II+III shows clear signs of blood clotting enzymes presence and activation by One-Stage assays, NaPTT, thrombin presence, PKA, “kallikrein” and TGT (generic & FXIdef) assays.

• Signs of activated factors (thrombin presence, PKA, “kallikrein” and TGT –both assays-) can be detected even after anion exchange purification (“kallikrein”, TGT FXIdef), including the acid pH treated intermediate.

• After Pasteurization there is not any indication of residual activated enzymes (negative NaPTT, thrombin presence, PKA, “kallikrein” and TGT –both assays-).

24

Study of Final Products

25

B2 10% C 10% E 10%

A 10%

Flebogamma®

5% DIFFlebogamma®

10% DIF

Flebogamma®

5%

A 5%

A 5% 2007

B1 5%

D 5%

0

50

100

150

200

250

300

350

Peak o

f th

rom

bin

acti

vit

y (

nM

)

Final products: FXI deficient plasma

TGT

NaPTT

Manufacturers:

A 5% 2007: n=1

A 5%: n= 3

A 10%: n=2-4

B1 5% (Lyophilised): n= 2

B2 10% (Liquid): n=2

C 10%: n=1

D 5%: n=1

E 10%: n=2

Flebogamma® 5%: n=5

Flebogamma® 5% DIF: n=4

Flebogamma® 10% DIF: n=5

A 10%

B2 10%

A 5%

( 2007)

A 5% B1 5%

C 10% D 5%

Flebogamma®

5%E 10%

Flebogamma®

10% DIFFlebogamma®

5% DIF

0

0.2

0.4

0.6

0.8

1

1.2

1.4

NaP

TT

sam

ple

/FX

I-D

ef

pla

sm

a r

ati

o

The lots tested were not among those published as involved in thromboembolic adverse events

26

Final products: Tests with PPP

NaPTT TGT

N Range (s)

Ratio

sample/control

(range)

N nM thrombin

(Mean ± SD)

A 5% (lot of 2007) 1 272 0.99 1 2

A 5% 3 173-217 0.62-0.78 3 168 ± 7

A 10% 1 202 0.73 3 172 ± 24

Flebogamma® 5% 2 314-315 1.21-1.21 4 -19 ± 47

Flebogamma® 5% DIF 3 233-269 0.82-0.97 3 -27 ± 9

Flebogamma® 10% DIF 3 242-272 0.92-0.97 1 6


27

FXI:C (IU/ml) FXI:Ag (Range; PEU/ml)1

Manufacturer A 5% 2007 (n=1) <0.007 0.005

Manufacturer A 5% 2010 (n=3) 0.08-0.10 0.09-0.12

Manufacturer A 10% (n=3 to 4) <0.007 0.004-0.009

Manufacturer B1 lyoph. (n=1 to 2) <0.007 0.003-0.006

Manufacturer B2 liq (n=2) NT 0.005-0.010

Manufacturer C (n=1) NT <0.003

Manufacturer D (n=1) NT <0.003

Manufacturer E (n=2) NT <0.003

Flebogamma® 5% DIF (n=4) <0.007 <0.003-0.008

Flebogamma® 10% DIF (n=4) <0.007 <0.003-0.025

Flebogamma® 5% (n=2) <0.007 <0.003-0.011

Final products

NT: Not Tested

1 3µg=1PEU (according to the mean of the results of 3 lots of human normal plasma in 11 different assays).


28

Final products: Additional One-stage tests

• FII, VII, IX, X, XII were not detected in

Flebogamma® DIF 5%, Flebogamma® 5%,

Manufacturer A 5% 2007, Manufacturer A 5% and

10% 2010.

• Manufacturer B1 showed also not detectable FVII

and FXII (other factors not tested).

• Other manufacturers not tested by One-Stage

assays.

29

Final Products: Summary

• Among all tested products, only products A and B1 show indications of coagulation activity markers, with high TGT & NaPTT (FXI-Deficient) values.

• Additionally, product A has high TGT-PPP and slightly shortened NaPTT-PPP times.

• Product A also shows FXI clotting activity by the one-stage clotting assay. The amount of FXI (at least partially activated) infused in an adult receiving 2g/kg would be aproximately 250 IU.


30

EFFECTIVENESS OF THE

ACID TREATMENT AND PASTEURISATION

TO ELIMINATE ACTIVATED FACTORS

31

MIX: POSITIVE

PROCESS MATERIAL

SAMPLE WITH ACTIVATED

FACTORS POSITIVE

5% 10% 20%

ACID TREATMENT (4.5 h 37ºC)

PASTEURIZATION (10h 60ºC)

AFTER ACID TREATMENT

POSITIVE

AFTER PASTEURIZATION

NEGATIVE

RESULTS

32

ACID TREATMENT: Limited effect on activated factors

Acid treatment 4.5h 37ºC (n=1)

Spike 1/20 Spike 1/10 Spike 1/5

Before After Before After Before After

NaPTT-PPP (s)

Neat 54 136 34 128 24 156

Diluted 1/5 119 225 82 213 58 231

Control (buffer) 288-293

PKA (IU/ml) 5 <2 24 <2 53 <2

"Kallikrein" (AU/min) 0.019 0.019 0.020 0.019 0.038 0.015

TGT FXI-Def. Plasma (nM) 286 239 323 263 331 267

FXI:Ag

PEU/ml1 0.25 0.26 0.34 0.31 0.22 0.20


33

Pasteurisation 10h 60ºC (n=1-3)

Spike 1/20 Spike 1/10 Spike 1/5

Before After Before After Before After

NaPTT-PPP (s)

Neat 53-133 269-291 36-99 255-294 24-77 255-293

Diluted 1/5 97-224 236-293 62-190 229-264 43-155 280-291

Control (buffer) 237-279

PKA (IU/ml) 4-28 <2 18-65 <2 26-113 <2

"Kallikrein" (AU/min) 0.010-0.011 0.001-0.002 0.012-0.014 0.000-0.002 0.015-0.019 0.000-0.002

TGT (nM)

PPP 81 -33 70 -42 89 -36

FXI-Def Plasma Mean±SD 387±72 40±37 419±64 30±24 456±55 18±18

FXI:Ag

PEU/ml1 0.21 0.06 0.30 0.02 0.45 <0.02

PASTEURISATION: marked effect on activated factors


34

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 9 10

20% Mixture (h)

Re

co

ve

ry (

%)

Peak of Thrombin activity (nM) PKA (IU/ml) "kalikrein like" (ΔAU/min)

Kinetics of clotting factor inactivation during

Pasteurization (20% v/v spike)

~90% reduction

TGT FXI-Def

Laboratory activated samples derived from FrII+III were

spiked into the industrial material before Pasteurization.

35

Pasteurization spiking experiments:

Conclusions

• After Pasteurization, samples with different degrees of activated factors

show :

– Disappearance of activated factors (NaPTT –both tests-, TGT –both

tests-, One-stage clotting factors, thrombin presence, PKA,

“kallikrein like”).

– Reduction of FXI:Ag.

– Levels of PKA lower than the detection limit after 2h of treatment.

– Relevant reduction of thrombin generation even after only 2h of

treatment.

Pasteurization* effectively inactivates clotting factors.

*: Under Grifols’ process conditions

36

• Grifols’ Pharmacovigilance Data

A search in the registry of the cases related to our IVIG products was performed

The most important terms from the “Standardised MedDRA* Queries” (“Embolic and thrombotic events”) were used

A new review of all potential TEEs was done by a revision of signs/symptoms of the adverse reactions notified to Grifols (again, from our registry) since the marketing authorisation of these products

The total amount of sales in grams of IVIGs over the world was used to express the number of TEE per million grams sold

Pharmacovigilance

* Medical Dictionary for Regulatory Activities

37

Pharmacovigilance

1: http://ec.europa.eu/health/documents/community-register/html/ho17601.htm#EndOfPage

Product Year(s) TEEs Report rate

(million g / case)

Octagam® (1) 2005 - 2007 1.49 – 1.60

Octagam® (1) 2008- 2009 0.51 – 0.46

Octagam® (1) Jan-Jul 2010 0.18

Flebogamma® Feb 1993 – Sep

2010 6.1

Flebogamma® DIF May 2007 – Sep

2010 6.0

pasteurisation inactivates coagulation enzymes … inactivates coagulation enzymes during flebogamma...

Documents