Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Characterization of Protein
Biopharmaceuticals
Koen Sandra
Webinar in association with SelectScience and Agilent
Technologies
January 28, 2015
Protein biopharmaceuticals
Therapeutic macromolecules produced via recombinant
DNA technology
Used in the treatment of life threatening diseases such
as cancer, autoimmune diseases, etc.
Global protein therapeutics market: $100 billion
(monoclonal antibodies + other recombinant proteins)
Protein biopharmaceuticals
Blockbuster protein biopharmaceuticals:
Protein characterization
Whether being involved in the development of
innovator biopharmaceuticals or biosimilars, an indepth characterization and analysis of these molecules
is required during their development and lifetime
Analysis is typically more challenging compared to small
molecule drugs
Proteins are large and heterogeneous
Protein characterization
Typical characteristics
EVQLVESGGGLVQPGGSLRLSCAAS
GFNIKDTYIHWVRQAPGKGLEW--
N-ter
N-ter
Hc
Lc
C-ter
C-ter
--NYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Protein characterization
Typical characteristics
EVQLVESGGGLVQPGGSLRLSCAAS
GFNIKDTYIHWVRQAPGKGLEW--
N-ter
N-ter
Hc
Lc
C-ter
C-ter
--NYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Protein characterization
Characteristics determined at different levels
Protein
Acid hydrolysis
Amino acids
Trypsin
digestion
Peptides
Sugars
N-glycans
Charge
Size
Hydro(phob/phil)icity
Affinity
CEX, AEX
SEC
RPLC, HIC, HILIC
Affinity Chromatography
Reversed-phase U/HPLC
Proteins
Reversed-phase U/HPLC
Challenges encountered in RPLC of proteins
Issue
Reason
Solution
Peak tailing
Secondary ionic
interactions
High number of pos
charges on proteins
Low Dm of large
molecules
Limited access to
pores
Widepore phases
Higher temperature
Efficient stationary
phase (sub 2 m,
superficially porous)
Hydrophobicity
Less hydrophobic
stationary phases
Stronger solvent
High temperatures
Peak broadening
Adsorption
Stationary phase
with limited access
to residual silanols
Ion-pairing reagent
Higher temperature
Courtesy of D. Guillarme
10
Reversed-phase U/HPLC
RPLC analysis for identity and purity determination of Herceptin
Hc
Lc
Hc
Fab
Lc
Fab
Fc
Intact
Fc
11
Deconvoluted spectra
12
Hc
f
mAU
120
Biosimilar
100
80
60
40
ab
20
0
mAU
10
12
14
300
16
18
16
18
20
min
Originator
250
200
150
100
50
0
10
12
14
20
min
13
LC + 2 Hex
23762.8
LC ox
23455.2
1
0
x10
LC + 1 Hex
23601.8
23439.4
4
2
0
x10
LC
1.5
23439.4
S
S
S
0.5
0
LC + deam
x10 4
1.5
1
0.5
23440.3
S
S
S
22600 22700 22800 22900 23000 23100 23200 23300 23400 23500 23600 23700 23800 23900 24000 24100 24200 24300
Counts vs. Deconvoluted Mass (amu)
14
x10 4
50724.3
2.5
2
1.5
1
C-ter
--NHYTQKSLSLSPGK
0.5
0
G0F
x10 5
3
50596.7
2.5
2
1.5
1
0.5
G1F
C-ter
50758.4
--NHYTQKSLSLSPG
0
x10 5
G1F
G0F
2.5
50758.6
50596.4
2
1.5
1
0.5
0
G0
G2F
50921.0
50450.7
50000 50100 50200 50300 50400 50500 50600 50700 50800 50900 51000 51100 51200 51300 51400 51500 51600
Counts vs. Deconvoluted Mass (amu)
15
Remicade
0.8
0.6
0.4
0.2
0
x10 2
6
Remicade
Remicade
biosimilar
4
2
0
0.5
1.5
2.5
3.5
4.5
5.5
6.5
7.5
8.5
Remicade
biosimilar
16
Remicade
0.8
0.6
0.4
0.2
0
x10 2
6
x10 3
Remicade
biosimilar
Remicade
1
0.9
Remicade
biosimilar
0.8
0.7
0
0.5
1.5
2.5
3.5
4.5
5.5
0.6
6.5
7.5
8.5
0.5
0.4
0.3
0.2
0.1
0
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
5.1
5.2
5.3
5.4
17
Blank
18
Hc
Lc
19
Ion exchange
chromatography
Proteins
20
+H N
3
S
+Na
+Na
+Na
mAU
A
G
800
Replicate 5
700
F
Y
P
25 cm x 2.1 mm x 5 Replicate
m Agilent Bio-mAb
1
Temp: 30C Flow: 200 L/min
Replicate 2
UV: 214 nm
Replicate
3
MPA: 10 mM phos pH
7.65
MPB: 10 mM phos pH
7.65,
100
mM
Replicate 4 NaCl
5-70%B, 0-36 min
+Lys
600
500
400
Asparagine
deamidation
300
200
Electrostatic interaction
100
between charged side chains
0
and opposite charged ion
exchange functionalities
Elution: increase salt
concentration or less common
pH
10
15
20
25
pI low
(ACIDIC)
30
35
min
pI high
(BASIC)
21
mAU
600
mAb with
one Lc deamidated
500
400
300
Non-stressed
originator
200
100
0
10
15
20
25
30
min
mAb with
mAb
one Lc deamidated
mAU
600
mAb with
both Lc deamidated
500
400
300
1 day pH 9 stressed
originator
200
100
0
10
15
20
25
30
min
mAb with
both Lc deamidated
mAU
600
500
400
300
200
3 days pH 9 stressed
originator
mAb
100
0
10
15
20
25
30
min
22
mAU
600
500
400
300
200
100
0
10
15
20
25
30
min
ND
23
Size exclusion
chromatography
Proteins
24
support
mAU
50
99.6%
Buffer related
compound
40
30
No interaction with
surface
Separation by means of
pores having different
accessibility for molecules
of different size
Elution with solvents that
suppress interactions with
column packing
20
0.4%
10
mAb
dimer
0
10
12
14
16
min
25
mAU
Originator
800
600
400
200
Buffer related
compound
mAb
dimer
0
0
10
12
14
16
min
mAU
Biosimilar
800
600
400
200
0
0
10
12
14
16
min
26
mAU
50
Buffer related
compound
Originator
40
30
20
mAb
dimer
10
0
0
10
12
14
16
mAU
50
min
Biosimilar
40
30
20
10
0
0
10
12
14
16
min
27
Reversed-phase U/HPLC
Peptides
28
29
Heavy Chain
A(1-449)
Light Chain
B(1-214)
EVQLVESGGGLVQPGGSLRLSCAAS
GFNIKDTYIHWVRQAPGKGLEW--
Hc
Lc
--NYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG
62 identity peptides
Modifications
Incomplete and aspecific cleavages
...
> 100 peptides
30
C18
O
F3 C C
O-
+H N
3
silica
A
G
F
Y
O
F3 C C
O-
P
+Lys
31
* *
T35
T5
T7
T11
T43
T46
T32
T34
T23
T42
T40
T18
T3
T41
T31
T29
T47
T24
T44 T53
T27
T19
T51
T38
T55
T59
T8
T45
T2
T1
T57
T15
T58
T22
T62
T41 ox
T16 T6
T21
T50
T54
T30
T3
T61
T56
T43
T22-23
T14
T3 deam
T37-38
T33
T25
T26 deam
T4
T12
T20
T36
T39
T48
T49
T45 + glycans
T26
T13
T10
T21 pyroGlu
32
33
peptides
Experimental MW
Mass spectrometry
Digestion
Peptide
ID
In-silico workflow
protein
peptides
In-silico
digestion (user defined)
Theoretical MW
In-silico
modifications (user defined)
34
35
Hc (A-chain)
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKG
RFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP
IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
Lc (B-chain)
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSG
TDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN
FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
TKSFNRGEC
36
99,02 %
T45 unglycosylated
0,98 %
T45 + glycans
T27
T19
T16
T6
T2
T47
T59
T24
T51
T8
T37-38
T38
T55
T45
unglycosylated
37
N-ter
N-ter
Hc
EEQYNSTYR
Lc
T45 + G0F
C-ter
C-ter
T45 + G1Fa
C-ter
T45 + G1Fb
C-ter
T45 + G0
T45 + G2F
38
N-ter
Hc
T43
ND
ND
Lc
C-ter
C-ter
T34
T23
MMox
T42
T18
T3
T41
+K
C-ter C-ter
T31
T13
T26
T25
T33
T50
T14
T21
T1
T22
T3 deam
T62 + K
T41 ox
T62
T26 deam
T26 deam
T29
T15
T3
T54
T30
T61
T56
39
Comparability assessment
Originator
Biosimilar
40
Comparability assessment
T45 + G0F
Originator
T45 + G1Fa
T45 + G1Fb
T45 + G2F
T45 + G0
T45 + G0F
Biosimilar
Undergalactosylation
observed in biosimilar
T45 + G1F
T45 + G0
41
Comparability assessment
ASQDVNTAVAWYQQKPGK
Originator
Biosimilar
T3 native
91,75 %
86,92 %
T3 deamidation
8,25 %
13,08 %
Originator
ASQDVDTAVAWYQQKPGK
ASQDVNTAVAWYQQKPGK
Biosimilar
ASQDVDTAVAWYQQKPGK
42
Deamidation
43
Comparability assessment
Originator
T62
Biosimilar
T62
--NHYTQKSLSLSPGK
T62+K
44
45
Originator 2
Clone (biosimilar)
46
MS Originator
T62
T62
T62+K
MS Biosimilar
T62+K
47
Hydrophilic interaction
chromatography
Glycans
48
PNGase F
2-AB labeling
LC-FLD (MS)
2-AB
Glycan profile
2-AB
Clean-up
Clean-up
2-Aminobenzamide (2-AB)
49
Glycan
LU
16
silica
14
G0F
1.8 m fully
porous particles
12
10
G1Fa
8
6
Glycan
G1Fb
G0
G2F
0
5
LU
16
Hydrophilic partitioning
between aqueous layer and
mobile phase
Elution: increase water
concentration
14
10
15
20
25
min
25
min
G0F
2.7 m superficially
porous particles
12
10
G1Fa
8
6
4
G1Fb
G0
G2F
0
5
10
15
20
50
G1Fa
1.8 m fully
porous particles
LU
G1Fb
G0-GlcNAc
2
1.5
1
0.5
G1-GlcNAc
2.5
G0F-GlcNAc
G0
G2F
G1F-GlcNAc
G1a
Man5
G1b
0
-0.5
8
10
12
14
G0F 16
18
20
LU
1.5
1
G0
0.5
G1-GlcNAc
G0F-GlcNAc
G0-GlcNAc
24
min
2.7 m superficially
porous particles
G1Fb
3
2.5
22
G1Fa
G2F
G1F-GlcNAc
G1a
Man5
0
-0.5
8
10
12
14
16
18
20
22
24
min
51
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
52
Comparability assessment
G0F
LU
17.5
Originator
15
12.5
G1Fa
10
7.5
5
2.5
G1Fb
G0
G2F
0
10
15
20
25
LU
30
min
Biosimilar
16
14
12
10
8
6
4
2
0
10
15
20
25
30
min
53
LU
15
12.5
10
7.5
5
2.5
0
G0
10
G1F
15
Biosimilar
G2F
20
25
30
min
LU
12
10
8
6
4
2
0
Biosimilar: 4x
10
15
20
LU
12
10
8
6
4
2
0
25
30
min
Biosimilar: 8x
10
15
20
25
30
min
LU
8
6
Biosimilar: 16x
4
2
10
15
20
25
30
min
LU
10
8
6
4
Biosimilar: 24x
2
0
15 of Protein Biopharmaceuticals
20
25
Strategies for the Separation and10Characterization
- Webinar30
min
54
Biosimilar
Biosimilar
4x
Biosimilar
8x
Biosimilar
16x
Biosimilar
24x
Specifications
originators
% G0-GlcNAC
0.55
0.49
0.47
0.53
0.61
% G0F-GlcNAc
5.35
2.72
2.15
1.78
1.67
% G0
1.57
2.67
1.98
2.35
2.02
% G1-GlcNAc
0.39
0.32
0.35
0.45
0.51
% G0F
70.80
48.91
46.39
44.19
43.72
44.39 +/-6.47
% Man5
8.22
5.79
4.95
6.21
7.14
% G1a + % G1F-GlcNAc
0.55
1.35
1.34
1.51
1.44
% G1b
0.17
0.32
0.29
0.37
0.38
% G1Fa
7.76
22.66
25.58
25.83
25.91
% G1Fb
3.62
8.11
8.70
8.75
8.62
% G1F
11.38
30.77
34.28
34.57
34.53
% G2F
1.01
6.66
7.80
8.03
7.98
55
Acknowledgement
Isabel Vandenheede, Emmie Dumont and Pat Sandra (RIC,
Kortrijk, Belgium)
The colleagues from the biopharmaceutical industry who
trigger and inspire us to develop and apply chromatographic
and mass spectrometric methodologies and strategies to
tackle their challenging requests
Maureen Joseph, Gina Goggins, Linda Lloyd, Phu Duong
(Agilent Technologies, Wilmington, Delaware)
www.richrom.com
koen.sandra@richrom.com
56
Confidentiality Label
January 28, 2015
57
Challenge
Reason
Solution
Peak
tailing
Lower
resolution
Less accuracy
Secondary ionic
interactions
High number of pos
charges on proteins
Lower
resolution
Reduced
sensitivity
Low Dm of large
molecules
Limited access to
pores
Widepore phases
Higher temperature
Efficient stationary phase
(sub 2 m, superficially
porous)
Poor recovery
Less sensitivity
Hydrophobicity
Peak
broadening
Adsorption
Column parameters are important to solve problems of efficiency/resolution and recovery for
improved LC and LC/MS characterization of proteins.
Confidentiality Label
January 28, 2015
58
0.25 um
450 pores
3.5 um
Superficially
porous
particle
AdvanceBio RP-mAb C4
AdvanceBio RP-mAb SB-C8
AdvanceBio RP-mAb Diphenyl
140
120
12
10
100
6
4
80
-2
60
-4
1.7
1.8
1.9
2.1
2.2
2.3
2.4
min
2.5
40
20
0
1.7
1.8
1.9
Method Parameters
Column dimensions: 2.1 x 100 mm
Mobile phase A: 0.1% TFA in water/IPA (98/2)
Mobile phase B: IPA/acetonitrile/MPA (70/20/10)
Flow rate: 1.0 mL/min
2.1
2.2
2.3
2.4
2.5
2.5
min
40 min.
Analysis
0.2mL/min
140 Bar
TIC
2.1 x 100mm, 2.7um
1.75
0.75
LC and LC/MS
0
1 2 3 4 5 6 7 8 9 101112131415161718192021222324252627282930313233343536373839
x108
4.6
3.6
0.50 um
14 min.
Analysis
0.6mL/min
433 Bar
2.6
1.7um
2.7um
1.6
120 pores
0.6
0
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.511 11.512 12.513
Confidentiality Label
January 28, 2015
61
3 m particle
Confidentiality Label
January 28, 2015
62
Protein
Efficiency
Gain
Thyroglobulin
2.2X
2460
1120
BSA Dimer
1.9X
5100
2720
BSA
2.0X
13090
6590
Ribonuclease A
2.0X
22000
11160
Uracil
1.4X
38500
27860
Peak
8
Min
10 11 12 13 14 15
Confidentiality Label
January 28, 2015
64
23.144
14.049
15-30% B in 30 min
6.906
100
4.782
0.856
1.360
1.688
1.969
2.100
2.243
2.649
2.937
3.211
300
200
Right- MAb-subspecies
15.901
16.565
400
o MAb, NP10
10
15
20
25
mAU
30
400
35
min
05-30% B in 30min
24.828
500
26.560
1.431
1.559
200
100
33.902
300
2.725
CX-10
MAb-subspecies-left
mAU
17.745
MAb-major
0
0
10
15
20
25
30
35
min
Characterization of mAb
65
1/28/2015
AdvanceBio
Glycan Mapping,
2.7 um
(superficially porous)
1260 Infinity
Bio-inert HPLC/FLD
+
1290 Infinity UHPLC
AdvanceBio
Glycan Mapping,
1.8 um
(totally porous)
66
Glycan analysis
Hydrophilic interaction chromatography (HILIC)
Charge variant analysis
Ion exchange chromatography (IEX)
Aggregation analysis
Size exclusion chromatography (SEC)
Agilent Technologies
January 28, 2015
67