Poly(ethylene glycol) chemistry and protein PEGylation

Poly(ethylene glycol) chemistry and protein PEGylation

Poly(ethylene glycol) chemistry and protein PEGylation
Sotir Zahariev, Corrado Guarnaccia, Ventzislav Zlatev and Sandor Pongor
Protein Structure and Bioinformatics Group, ICGEB Trieste
G-CSF PEGylation using a linear 20KDa mPEG-CHO derivative
RP-HPLC and SDS-PAGE pegylation reaction analysis
Chromatogram
100
2
mPEG-GCSF
400
G-CSF
50
mVolts
In recent years, poly(ethylene glycol) (PEG) conjugation (PEGylation) have been
shown to confer superior clinical useful properties to proteins of therapeutic interest such
as better physical and thermal stability, greater protection against proteolytic degradation,
higher solubility, longer in vivo circulating half-lives, and lower clearance, thus enhancing
efficacy. Additional qualities of pegylated proteins are reduced immunogenicity and
antigenicity, as well as reduced toxicity.
To couple a monofunctional PEG (mPEG) moiety to a protein it is first necessary to
activate the polymer by converting the hydroxyl terminus to some functional group capable
of reacting with the functional groups found on the surface of proteins. The most common
method has been to activate the PEG moiety with functional groups suitable for reaction
with protein amino groups.
To evaluate the potential of different “activation” chemistries we have conducted
comparative studies of synthesis of linear and branched mPEG derivatives with various
MW’s, developed novel chromatographic methods for their purification and tested some of
these compounds for the PEGylation of interferon alpha 2a (IFN2a) and of
granulocyte colony stimulating factor (G-CSF)
% Mobile Phase
Introduction
multimers
mPEG-GCSF
200
G-CSF
multimers
3
1
0
Polyethylene glycol chemistry
0
0
20
40
Minutes
We developed novel chemical methods for the preparation of various mPEG molecules
for conjugation:
> C:\DOCUME~1\guarna\Desktop\GILSON~1\GCSF_P~2\GCSF.067\GCSF.GDT : UV Channel 1 : mPEG20-CHO + GCSF 2500mg/350mg (7:1), O/N from 3rd 1100mg CHO addition , ZorbaxCN, 214nm: Inj. Number: 1
Pump 1
Pump 2
MALDI-TOF pos. linear mode of the main fraction
from RP-HPLC
 mPEG active esters (4-nitrophenyl-, hydroxysuccinimidyl-, benzotriazolyl-) with MW
from 3000 to 30000
4700 Linear Spec #1 MC=>NR(2.00)=>NR(2.00)=>NF0.7=>SM21[BP = 9998.5, 3821]
13961.8643
14225.1895
 branched-PEG (based on diamino-carboxylic acids) with MW from 10000 to 40000Da
 Purification of these branched-PEG by various chromatographic techniques and their
transformation in hydroxysuccinimidyl esters.
14586.2334
30
1146.4
14839.9531
15411.2354
15039.4258
15116.9941
15651.3760
mPEG-GCSF: 41460.1328 (MH+)
15723.9775
20575.0078
17571.1660
20483.0410
16437.4766
21051.8809
17686.9551
21344.1504
18424.3770
19345.9707
 protected mPEG-aldehydes (MW=3000-30000)
18485.7813
22378.6953
22148.5410
% Intensity
22829.8711
 mPEG-carboxylates (MW 3000 -30000): mPEG-OX (X= (CH2)nCOOH; n=0-3
24138.4980
24248.7617
25536.3242
26433.9961
27359.3672
20
41460.1328
29609.5391
31298.8359
39338.2422
34682.5313
35669.5039
38365.6602
43753.8516
IFN Alpha 2a PEGylation using a branched 40 KDa mPEG-COOSu derivative
44167.5781
51694.6523
Reaction parameters: mPEG-COOSu stability/activity, mPEG excess, time, temperature, pH
to minimize multimers and preserve biological activity of both mono-PEG-IFN and nonreacted-IFN.
1 00
Chromat ogram
40
10
9998.0
4
3
3
m V o lt s
% M o b ile P h a s e
20
50
HiTrap S P of 1 1 mg p eg ylation mix of G CS F:10 _U V1_280 nm
HiTrap S P of 1 1 mg p eg ylation mix of G CS F:10 _Cond
Area %
- tri-PEG-IFN 1
mAU
2071.39
0.11
- di-PEG-IFN 2
25 00
15.98
2
17.10
174078.08
9.07
3
18.03
783321.62
40.80
4
19.13
931500.62
48.52
5
19.98
24538.91
1.28
6
22.58
4306.66
0.22
A rea
28076.8
37116.2
46155.6
55195.0
mPEG-GCSF purification and quality control
1
Peak R. Time
19037.4
Mass (m/z)
RP-HPLC reaction monitoring
4
47231.7266
47608.5469
HiTrap S P o f 11 mg peg ylation mix of G CSF:1 0_U V 2_220n m
H iTrap SP of 11 mg pegylatio n mix o f GCS F:10_ UV3_ 214nm
H iTrap SP o f 11 mg peg ylation mix of G CSF :1 0_Conc
H iTrap S P of 11 mg pegylation mix of GCS F:10_F ractions
HiTrap S P of 1 1 mg p eg ylation mix of G CSF :1 0_Lo gbook
- mono-PEG-IFN 3
20 00
-mPEG(40KDa)
(UV transparent)
2
2
15 00
6
1
5
1
0
10 00
0
0
20
M in utes
mPEG-GCSF
- IFN 4
40
5 00
Mono mPEG-IFN alpha2a purification and quality control
0
A1
40
60
80
10 0
A2
A3
A4
A5
1 20
Was te
1 40
1 60
ml
Std PEG-IFN
CAM B IF N 47 mg P egy la ti o n mix CM p urification :Rec ov ered_ UV1_ 28 0n m
CAM B IF N 47 mg P egy la ti o n mix CM p urification :Rec ov ered_ pH
CAM B IF N 47 mg P egy la ti o n mix CM p urification:Rec ove red_ Con d
CAMB IFN 4 7mg Peg ylation m ix CM pu rifica ti o n:Reco vered _F raction s
CAM B IF N 47 mg P egy la ti o n mix CM p urification :Rec ov ered_ Con c
CAM B IF N 47 mg P egy la ti o n mix CM p urification:Rec ove red_ Lo gb ook
mAU
Std PEG-IFN
Mono-PEG-IFN
30 0
1.
MW marker
2.
empty
3.
GCSF
4.
frac A1
5.
frac A2
6.
frac A3
7.
frac A4
8.
frac A5
9.
empty
10. empty
25 0
250KDa150KDa100KDa75KDa-
20 0
15 0
Pure Fractions
50KDa37KDa-
IFN
multi-PEG-IFN
10 0
Fractions from the purification
25KDa-
50
(c)
0
X1
300
X2
X3
X4
4 00
Waste
A 1 A 3 A 5 A 7 A 9 A11 B12 B10 B8 B6 B4 B2 C1 C3 C5 C7 C9 C11 D12
5 00
6 00
7 00
8% SDS-PAGE
Coomassie staining
Waste
8 00
ml
(a)
Gradient 4-12% SDS-PAGE
Barium staining (PEG specific)
(b)
Fig.1
Gradient 4-12% SDS-PAGE
Barium staining (PEG specific)
Gradient 4-12% SDS-PAGE
Coomassie staining
The mono-pegylated IFN alpha 2a is purified by LC (Fig. 1a) and the fractions
checked by SDS-PAGE (Barium staining and Coomassie staining, Fig. 1b and 1c)
MALDI-TOF analysis of a trypsin digest of 20 ug aliquots of GCSF and mPEG20K-GCSF
demonstrates that the site of mPEG attachment is the N-terminus of the molecule
Final - Shots 600 - 6475_28-08-07; Label F11
(a)
Circular dichroism
(b)
61796.9
LysC peptide mapping
450
(c)
100
2032.9764(R12236,S1998)
2.1E+4
80
350
MALDI-TOF
60
250
mPEG20K-GCSF
40
150
20
1607.8749(R12461,S57)
1785.0464(R15122,S30)
50
2054.9409(R13958,S365)
2232.9983(R14180,S192)
2780.3208(R9593,S 58)
0
1600
1880
2160
2440
2720
3000
-50
-IFN before
pegylation
-mPEG-IFN
rHuMet-GCSF 1-17 (MTPLGPASSLPQSFLLK)
molecular mass monoisotopic (MH+):
1786.97
-150
-250
mPEG-IFN
1.7E+4
1786.9087(R11617,S1188)
-350
2189.0757(R11451,S1427)
80
0
Fig. 2
Accumulated - Shots 600 - 6475_28-08-07; Label F10
2032.9847(R11230,S1521)
100
Std-PEG-IFN
Mass (m/z)
30
time (min)
60
60
GCSF
40
20
(d)
45
25
5
-15
-35
m PEG-IFN Pool
Pegasys
-55
0
-75
10
20
30
40
50
60
70
The quality controls confirm the purity and
quality of the monopegylated IFN also in
comparison to a standard of pegylated IFN:
RP-HPLC (not shown), CD (Fig. 2a), MALDITOF (Fig.2b), peptide mapping (Fig. 2c),
positional isomers composition (Fig. 2d)
The in vitro bioassay returns bioactivity values
perfectly in range with the values of standard
pegylated IFN (~3% of non-pegylated IFN).
1808.8917(R14499,S180)
2211.0566(R14944,S174)
2017.9810(R11893,S26)
2505.2896(R17723,S22)
2780.2695(R24385,S90)
0
1600
1880
2160
2440
2720
3000
Mass (m/z)
Conclusions
We established scalable protocols for the synthesis of activated linear and branched mPEG
derivatives with good purity and yields. The activated derivatives were efficient in tests of
pegylation of IFN alpha 2a and G-CSF giving monopegylated molecules with caracteristics
comparable to commercial standards.