pasteurisation inactivates coagulation enzymes … inactivates coagulation enzymes during flebogamma...
TRANSCRIPT
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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
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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
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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
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• 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
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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
14
15
PASTEURIZATION INACTIVATES SERINE PROTEASES
(Clotting proenzymes and Complement components are serine proteases)
High serine protease activity
Purification process
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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.
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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.
19
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).
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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
)
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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-).
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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
The lots tested were not among those published as involved in thromboembolic adverse events
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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).
The lots tested were not among those published as involved in thromboembolic adverse events
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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.
The lots tested were not among those published as involved in thromboembolic adverse events
30
EFFECTIVENESS OF THE
ACID TREATMENT AND PASTEURISATION
TO ELIMINATE ACTIVATED FACTORS
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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
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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
1 3µg=1PEU (according to the mean of the results of 3 lots of human normal plasma in 11 different assays).
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
1 3µg=1PEU (according to the mean of the results of 3 lots of human normal plasma in 11 different assays).
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