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