characterisation of recombinant fviii products · 2018. 4. 2. · a1 a2 a2 a3 c1a3 c2 b 1 372 1639...
TRANSCRIPT
Characterisation of recombinant FVIII
products Per F. Nielsen, Novo Nordisk A/S
6/20/2014 1
6/20/2014 2
• Coagulation FVIII in general
• Deamidation hot spots (relevant for potential liquid formulation)
• Sulfation at Tyrosine-1680
• Methionine oxidation
• “Degradation” forms in rFVIII products
Outline
6/20/2014 3
• FVIII (antihaemophilic factor)
• 2351 amino acids, 2332 amino acids after secretion
• Large, complex glycoprotein of approximately 300 kD
• In coagulation, FVIII acts as a cofactor to accelerate cleavage of FX
to FXa by FIXa on a phospholipid surface (platelet)
• FVIII dramatically increases maximal velocity of reaction
Coagulation factor VIII
6/20/2014 4
Primary structure
• 2332 amino acids (aa)
• Three A-domains
• One B-domain
• Two C-domains
Circulating FVIII
• Cleaved during secretion
• Circulates as a two-chain heterodimer
• Heavy chain (HC) and light chain (LC) are associated by metal ion-dependent binding
740 aa 908 aa 684 aa
C2 C1 A3 B A2 A1
Clevage during secretion
A2 A1 HC
LC
372 aa 740 aa
B
C2 C1 A3
1689 aa
Coagulation factor VIII
Expressed in Construct Protein
Turoctocog alfa CHO 1445 aa B-domain truncated
Product 1 CHO 1438 aa B-domain deleted
Product 2 CHO 2332 aa “Full length”
Product 3 BHK 2332 aa “Full length”
rFVIII preparations used
6/20/2014 5
CHO = Chinese Hamster Ovary; BHK = baby hamster kidney cells
Presentation title Date 6
Asp residues (highlighted in yellow): 82 Asn residues (highlighted in green): 63
1 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITD D D N D N D
88 LKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGN DD DD N D D D N
175 ALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGD N D D N N
262 TTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDN D D D NN DYDD
349 DLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETD D D DDDN D D DD NN D
436 FKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTD N N D D D
523 KSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQD N D D N D N D N N N D
610 ASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDN N D D D N N D
697 FRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSQNPPVLKRHQR-EITRTTLQSDQEEIDYDDTISN D N DYY D Y D NN N N D DYDD
784 VEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLD D YD D N D N D N
871 GLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLDN N D N N D D D D
958 EKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPD N N D N N N D N N D
1045 GLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYD N N N
1132 SNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMN D N D
1219 YSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITD N N D N N D N D
1306 ASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGN N NN D D N
1393 KVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYN D N D D
Turoctocog alfa – amino acid sequence
Deamidation/IsoAsp formation
• Should be adressed with respect to potential liquid formulation
• LC-MS of trypsin digest used for detection/quantification – due to the complexity of rFVIII it is difficult to monitor deamidation at the intact protein level
• Forced deamidation study with turoctocog alfa identified 6 potential deamidation sites (Five N-G and one N-A - all surface exposed)
• One site only detected following deglycosylation and one site only detected using alternative proteolytic enzyme
• Sample preparation performed at pH~6 and room temperature in order to lower the risk of inducing deamidation during handling
Dipold K et al. PLoS ONE 2012, 7(1) e30295
Asn and Asp degradation pathway
Presentation title Date 8
-17 Da -18 Da
+1 Da
+14 Da
Typically, succinimide, formed either by deamidation of asparagine or de-hydrolysis of Asp, is a short-lived intermediate and is rapidly hydrolyzed to Asp/iso-Asp. However, it is stable at acidic pH.
• Asp IsoAsp <<< Asn Asp/IsoAsp (at physiological pH)
• RP-HPLC elution: Asn<Asp<IsoAsp
• Asn-Gly 10-50x faster than Asn-Ala/Ser/Leu
Deamidation analysis
Presentation title Date 9
N8-GP poolede henstand trypsin TCEP
Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
%
4
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
%
0
PFN05112013-10 1: TOF MS ES+ 588_590
1.71e4
8.65
18.309.46
PFN05112013-10 1: TOF MS ES+ TIC
1.72e5
1.1215.29
8.657.32
6.61
1.96
2.37 2.51 3.50 6.07
5.14 5.66
8.1914.92
14.7611.369.26
9.44
10.97
10.08
12.88
12.2514.2013.17
18.38
18.09
16.72
16.0717.17
17.35
18.84
19.51
19.89
Asn
Iso-Asp Asp
AA409-418: SQYLNNGPQR
Presentation title Date 10
Deamidation level (%)
Stressed sample**
Turoctocog alfa
Product 1 Product 2 Product 3 PD FVIII
Asn-144 (NG) 50 <1 <1 <1 <1 <1
Asn-235* (NG) 100 <1 <1 <1 <1 ND
Asn-414 (NG) 100 <1 1 <1 <1 <1
Asn-1922 (NG) 90 <1 <1 <1 <1 ND
Asn-2060 (NA) 70 <1 <1 <1 <1 ND
Asn-2277 (NG) 100 <1 5 7 2 ND
*Asn-235 is covered following PNGaseF treatment. ** high pH, 37C
0
5
10
15
20
25
Asn-144 (HC) Asn-414 (HC) Asn-274 (LC) Asn-629 (LC)
Deamidation 12 weeks
pH6.5 5C
pH6.9 5C
pH6.5 25C
pH6.9 25C
Asn-414(HC)/Asn-414: Charge change acceptable, far from FIX binding region, exposed Asn-629(LC)/Asn-2277: Charge change acceptable, C2 domain, exposed
pH 6.5 and 6.9 at 5o and 25
oC
Deamidation varies with pH and temperature
% d
eam
idation
• 43 methionine residues present in rFVIII
• Monitored at intact protein level by RP-HPLC (Met-2199)
• Forced oxidation has etablished the following methionine residues as most prone to oxidation: Met-17, 337, 539, 1672, 1973, 2199
• No significant Met oxidation detected in the rFVIII products
Met oxidation
6/20/2014 12
H2O2
LC
HC
• FVIII is sulfated at six different Tyrosine residues
• Sulfation at Tyr-1680 important for vWF binding
• Monitoring of sulfation at Tyr-1680 based on peptide-map using capillary column chromatography and high resolution MS
Sulfation at Tyr-1680
6/20/2014 13
A2 A1 a2
C2 C1 A3 a3
B
1 372
1639
a1
740 761
1648 2020 2173 2332
SulfoTyr718
SulfoTyr719
SulfoTyr723
SulfoTyr1664 SulfoTyr1680
SulfoTyr346
HC
LC
Leyte A et al. J Biol Chem 1991; 266: 740-746, Michnick DA et al., J Biol Chem 1994; 269: 20095-20102
Sulfated and non-sulfated KEDFDIYDEDENQSPR can be separated on RP-HPLC C-18 column (0.1%TFA/Acetonitril gradient)
min 6 8 10 12 14 16 18 20 22 24
mAU
0
200
400
600
800
1000
DAD1 A, Sig=214,4 Ref=450,80 (091002\SIG11276.D)
14.8
23
15.2
44
15.7
77
min 6 8 10 12 14 16 18 20 22 24
mAU
0
200
400
600
800
1000
1200
1400
DAD1 A, Sig=214,4 Ref=450,80 (091002\SIG11277.D)
14.8
24
15.0
86
15.7
77
min 6 8 10 12 14 16 18 20 22 24
mAU
0
100
200
300
400
DAD1 A, Sig=214,4 Ref=450,80 (091002\SIG11272.D)
15.2
62
min 6 8 10 12 14 16 18 20 22 24
mAU
0
250
500
750
1000
1250
1500
1750
DAD1 A, Sig=214,4 Ref=450,80 (091002\SIG11273.D)
14.7
87
15.0
83
15.7
69
17.0
71
min 6 8 10 12 14 16 18 20 22 24
mAU
0
200
400
600
800
1000
1200
1400
DAD1 A, Sig=214,4 Ref=450,80 (091002\SIG11277.D)
14.8
24
15.7
77
Overnight incubation at low pH does not lead to loss of sulfate
1 Lys-Glu-Asp-Phe-Asp-Ile-Tyr-Asp-Glu-Asp-Glu-Asn-Gln-Ser-Pro-Arg-Tyr-
AA 1674-1689
Synthetic peptides +/- sulfated tyrosine
*
*
*
*
*: tBu impurity
Mix
Sulfated peptide
Non-sulfated peptide
pH~2 ON
6/20/2014 15
In-source fragmentation
RT: 14.97 - 30.02
16 18 20 22 24 26 28 30
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
Rela
tive A
bundance
23.92
23.89
23.95
24.5123.7723.19 25.28 26.30 27.49 28.2721.7819.9619.1518.4116.5114.98
23.28
23.30
23.32
23.3323.23
23.35
23.9522.89 24.3122.27 25.95 27.17 28.6119.1616.06 16.82 17.83
NL: 1.12E9
m/z= 1040.40-1040.42 F: FTMS + p NSI Full ms [300.00-2000.00] MS PFN_N8_Sample8
NL: 5.93E8
m/z= 1000.42-1000.44 F: FTMS + p NSI Full ms [300.00-2000.00] MS PFN_N8_Sample8
PFN_N8_Sample8 #5257-5269 RT: 23.92-23.97 AV: 13 NL: 6.47E8T: FTMS + p NSI Full ms [300.00-2000.00]
400 600 800 1000 1200 1400 1600 1800 2000
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
1040.91
667.62
701.601000.92530.44 711.91
1387.54
1051.40
1248.89843.51391.28 1560.86661.95 1843.86
1118.50
1665.65 1973.29
PFN_N8_Sample8 #5106-5126 RT: 23.24-23.33 AV: 21 NL: 1.97E9T: FTMS + p NSI Full ms [300.00-2000.00]
400 600 800 1000 1200 1400 1600 1800 2000
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
667.63
1000.93
800.95510.21
674.95
811.53
983.39
1334.241200.72
652.62
1011.93445.12 1500.64 1667.55 1818.02 1907.71
2+
3+
3+
2+
EIC: Sulfated peptide
EIC: Non-sulfated peptide
Quantification of sulfation using all charge-states
Sulfation at Tyr-1680: summary
6/20/2014 16
Expressed in
Non- sulfated Tyr-1680
Turoctocog alfa CHO <0.5%
Product 1 CHO 3-5%*
Product 2 CHO 4-10%*
Product 3 BHK 0.5-1.5*
pdFVIII <0.5%
Nielsen PF et al. Haemophilia 2012;18:e397-8; Kannicht C et al. Throm Res 2013; 131:78-88
*Batch-to-batch variation
Example of analysis of Tyr-1680 sulfation using the intensities of the isotopes from high-resolution MS analysis for quantification. Isotopic distribution of the doubly charged molecular ions of the tryptic peptide containing Tyr-1680 are depicted to the left, while the corresponding ion intensities are shown to the right
• SEC-MALS analysis of products
• Preparative RP-HPLC used for isolation of components
• Isolated fractions characterised by
• MALDI-MS and LC-MS
• Peptide mass fingerprinting
• N-terminal sequencing
• Analytical RP/SE HPLC
Structural analysis of main components in rFVIII products
6/20/2014 17
Structure of rFVIII products
Protein Characterizaion, Biopharm (634.02)
Slide no 18 Date
rFVIII product Expected MW* (kDa)
Product 1 ~174
Product 2 ~315
Product 3 ~315
Turoctocog alfa ~176
* of main component including glycosylation
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 44.0 46.0 48.0 50.0 52.0 54.0 56.0 58.0 60.0-1.4
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.5HPLCD_20140527 #5 N8 [modif ied by HeHo] NN7008 AC2N8Z4012 40ug 212513881 UV_VIS_1mAU
min
Dim
er -
20.
920
Mon
omer
- 2
2.81
3
Pos
tmon
omer
- 2
5.29
3
WVL:280 nm
19 Protein Characterizaion, Biopharm (634.02) Date
~MW (kDa)
HMWP2 HMWP1 Dimer/Pre-monomer
Monomer Post-monomer
2611 500 148 164 138
~MW (kDa)
HMWP Dimer Monomer LMWP1 LMWP2 LMWP3 LMWP4 LMWP5
1630 300 232 164 153 182 123
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 44.0 46.0 48.0 50.0 52.0 54.0 56.0 58.0 60.0-5.0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0HPLCD_20140527 #10 N8 - unknow n peaks [modif ied by HeHo] Advate 40ug 212513899 UV_VIS_1mAU
min
1 -
15.1
27
2 -
16.2
27
3 -
16.7
07
4 -
18.3
27
5 -
19.8
93
6 -
21.4
27
7 -
23.0
33
-
25.2
53
WVL:280 nm
Product 2
Turoctocog alfa
SEC-MALS comparison of rFVIII products
SE-HPLC
6/20/2014 20
Product 2 Full length
Turoctocog alfa
Jankowski et.al. reported characterisation of rFVIII forms isolated by size exclusion chromatography
Jankowski MA et al. Haemophilia 2007; 266: 30-37
RP-HPLC
Presentation title Date 21
Product 3 Full length
Product 2 Full length
Product 1
Turoctocog alfa
LC
HC
Presentation title Date 22
Example of analysis of isolated RP-HPLC fraction
A3 C1 C2
1314
Cycle PTH-aa pmol Theory
AA1314-
1 Ala 3 Ala
2 Leu 3 Leu
3 Lys 3 Lys
4 Gln 3 Gln
5 Phe 3 Phe
127786.293
64145.017
82896.728
43241.386
Advate Peak 1 from RP HPLC _ZipTip_ADNi 0:A1 MS Raw
0
250
500
750
1000
1250
1500
Inte
ns. [a
.u.]
25000 50000 75000 100000 125000 150000 175000 200000 225000 250000m/z
Direct MALDI-MS (peak 1) Edman sequencing (peak 1)
16
80
.90
7
12
91
.72
7
15
84
.91
6
20
33
.96
9
13
62
.58
8
19
83
.89
7
17
96
.85
2
16
35
.94
7
14
43
.72
6
25
21
.38
5
11
92
.49
4
11
05
.49
7
14
93
.73
0
35
78
.63
3
Advate Peak 1 RP HPLC Trp_DTT ADNi
0
250
500
750
1000
1250
1500
Inte
ns. [a
.u.]
1000 1500 2000 2500 3000 3500m/z
Peptide mass finger-printing (peak 1)
Product 2
6/20/2014 23
Major FVIII structures detected
Comments
Product 1 1-720 1-740 1649-2332 (LC)
Deamidation Asn-2277: <1% Tyr-1680 sulfation:95-98%
Product 2 (full length)
1-1313 1-817 1-740 (HC) 1314-2332 1649-2332 (LC) 1658-2332
Free LC ~5% (SE-HPLC) 1314-2332 ~16% (RP-HPLC) Deamidation Asn-2277: 5-10% Tyr-1680 sulfation: 90-95%
Product 3 (full length)
1-1313 1-817 1-740 1649-2332 (LC) 1658-2332
Free LC ~5% (SE-HPLC) Tyr-5 sulfation detected Deamidation Asn-2277: <1% Tyr-1680 sulfation: >98%
Jankowski MA et al. Haemophilia 2007; 266: 30-37
• Full length rFVIII product displays higher heterogeneity than B-domain truncated/deleted products. Main heterogeneity arise from B-domain truncations and free light chain
• rFVIII products display various levels of sulfation at Tyr-1680 with turoctocog alfa being fully sulfated
• Potential deamidation hotspots have been identified and low levels of deamidation can be detected in some of the rFVIII products (freeze dried)
Summary
24 Protein Characterizaion, Biopharm (634.02) Date