Upload
lexuyen
View
223
Download
0
Embed Size (px)
Citation preview
Strategies for the Separation and Characterization of Protein Biopharmaceuticals
Koen SandraWebinar in association with SelectScience and Agilent TechnologiesJanuary 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)
• ±20% of the total pharmaceutical market• Within the current decade, more than 50% of new drug approvals will be biologics
2Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Protein biopharmaceuticals
• Blockbuster protein biopharmaceuticals:– Insulin: Lantus (Sanofi‐Aventis)
– EPO: Epogen/Aranesp (Amgen)
– Trastuzumab: Herceptin (Roche/Genentech)– Infliximab: Remicade (J&J)
– Adalimumab: Humira (AbbVie)
– Etanercept: Enbrel (Pfizer, Amgen)
– …
• Several of these blockbusters are, or will soon become, open to the market
• This has resulted in an explosion of biosimilar activities
3Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Protein characterization
• Whether being involved in the development of innovator biopharmaceuticals or biosimilars, an in‐depth 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
4Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
• Typical characteristics– Amino acid sequence– Amino acid composition– Structural integrity – Higher order structures– Aggregation– S‐S bridges– N‐ and O‐glycosylation– N‐ and C‐terminal sequence– Charge variants– Deamidation/isomerization– Oxidation– Clipping– …
Protein characterization
5Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
--NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW--
C-ter C-ter
N-terN-ter
Hc
Lc
• Typical characteristics– Amino acid sequence– Amino acid composition– Structural integrity – Higher order structures– Aggregation– S‐S bridges– N‐ and O‐glycosylation– N‐ and C‐terminal sequence– Charge variants– Deamidation/isomerization– Oxidation– Clipping– …
Protein characterization
6Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
--NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW--
C-ter C-ter
N-terN-ter
Hc
Lc
• Characteristics determined at different levels
Protein characterization
7Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Protein
Peptides Sugars
Amino acid composition Amino acid sequence, modifications, modification sites, disulfide bridges, etc.
N‐glycans
Trypsindigestion
PNGase FAcid hydrolysis
Amino acids
MW, structural integrity, charge variants, aggregation, modifications
A wide range of separation modes
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 8
Charge CEX, AEXSize SECHydro(phob/phil)icity RPLC, HIC, HILICAffinity Affinity Chromatography
Reversed‐phase U/HPLC
9Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Proteins
• Challenges encountered in RPLC of proteins
Reversed‐phase U/HPLC
10Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Issue Reason Solution
Peak tailing • Secondary ionicinteractions
• High number of pos charges on proteins
• Stationary phasewith limited access to residual silanols
• Ion‐pairing reagent• Higher temperature
Peak broadening • Low Dm of large molecules
• Limited access topores
• Widepore phases• Higher temperature• Efficient stationary
phase (sub 2 µm, superficially porous)
Adsorption • Hydrophobicity • Less hydrophobicstationary phases
• Stronger solvent• High temperatures
Courtesy of D. Guillarme
Reversed‐phase U/HPLC
11Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
LcHc
Fc
Fab
Intact
HcLc
Fab
Fc
10 cm x 2.1 mm x 1.8 µm Zorbax 300 SB-C8Temp: 80°C Flow: 200 µL/min UV: 214 nmSolv A: 0.1% TFASolv B: 0.1% TFA in ACN 30-38.6%B, 2-25 min
RPLC analysis for identity and purity determination of Herceptin
Reversed‐phase U/HPLC – Mass Spectrometry
12Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
RPLC-UV-MS of Herceptin Lc and Hc Deconvoluted spectra
Reversed‐phase U/HPLC for comparability assessment
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 13
min8 10 12 14 16 18 20
mAU
0
20
40
60
80
100
120
min8 10 12 14 16 18 20
mAU
0
50
100
150
200
250
300
Biosimilar
Originator
Lc Hc
e
f
ab
c
d
g
Reversed‐phase U/HPLC for comparability assessment
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 14
Reversed‐phase U/HPLC for comparability assessment
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 15
G0F + C‐terminal Lys
Undergalactosylation observed in biosimilar
3x10
0
0.2
0.4
0.6
0.8
1
2x10
0
2
4
6
Response Units vs. Acquisition Time (min)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
Widepore Poroshell for comparability assessment
16Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Remicade
Remicade biosimilar
Advance Bio RP-mAb5 cm x 4.6 mm x 3.5 µm 450Å C4Temp: 80°C Flow: 1 mL/min UV: 214 nmSolv A: 0.1% TFA Solv B: 0.1% TFA in 90% ACN30-42.5%B, 0-6 min
1x10
00.5
11.5
22.5
33.5
44.5
1x10
00.5
11.5
22.5
33.5
44.5
Response Units vs. Acquisition Time (min)2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6
Remicade
Remicade biosimilar
3x10
0
0.2
0.4
0.6
0.8
1
2x10
0
2
4
6
Response Units vs. Acquisition Time (min)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
Widepore Poroshell for comparability assessment
17Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Differences in hydrophobicity due to a 2-point mutation in the AA sequence of the biosimilar
Advance Bio RP-mAb5 cm x 4.6 mm x 3.5 µm 450Å C4Temp: 80°C Flow: 1 mL/min UV: 214 nmSolv A: 0.1% TFA Solv B: 0.1% TFA in 90% ACN30-42.5%B, 0-6 min
Remicade
Remicade biosimilar
3x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Response Units vs. Acquisition Time (min)
4 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5 5.1 5.2 5.3 5.4
Remicade
Remicade biosimilar
Widepore Poroshell – zero carry‐over
18Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
1x10
00.20.40.60.8
11.21.41.61.8
22.22.42.62.8
33.23.43.63.8
44.2
Response Units vs. Acquisition Time (min)3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6
Advance Bio RP-mAb5 cm x 4.6 mm x 3.5 µm 450Å C4Temp: 80°C Flow: 1 mL/min UV: 214 nmSolv A: 0.1% TFA Solv B: 0.1% TFA in 90% ACN30-42.5%B, 0-6 min
Remicade(4 µg o.c.)
Blank
Remicade is a mAb which is prone to carry-over on a substantial number of RPLC columns → No carry-over observed on Widepore Poroshell column
Widepore Poroshell for comparability assessment
19Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Differences in hydrophobicity due to a 2-point mutation in the AA sequence of the biosimilarcompared
Advance Bio RP-mAb5 cm x 4.6 mm x 3.5 µm 450Å C4Temp: 80°C Flow: 1 mL/min UV: 214 nmSolv A: 0.1% TFA Solv B: 0.1% TFA in 90% ACN30-42.5%B, 0-6 min
RemicadeRemicade biosimilar Lc
Hc
Ion exchange chromatography
20Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Proteins
Ion exchange chromatography
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 21
WCX analysis (n=5) of Herceptin highlighting charge variants
• Electrostatic interaction between charged side chains and opposite charged ion exchange functionalities• Elution: increase salt concentration or less common pH
pI high(BASIC)
pI low(ACIDIC)
min5 10 15 20 25 30 35
mAU
0
100
200
300
400
500
600
700
800
Replicate 1Replicate 2Replicate 3Replicate 4Replicate 5
Asparagine deamidation
25 cm x 2.1 mm x 5 µm Agilent Bio-mAbTemp: 30°C Flow: 200 µL/min UV: 214 nmMPA: 10 mM phos pH 7.65MPB: 10 mM phos pH 7.65, 100 mM NaCl5-70%B, 0-36 min
+H3N
+Lys
S
A
G
F
Y
P
_
_
_
_
_
+Na
+Na
+Na
min10 15 20 25 30
mAU
0100200300400500600
min10 15 20 25 30
mAU
0100200300400500600
min10 15 20 25 30
mAU
0100200300400500600
Ion exchange chromatography
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 22
WCX analysis of stressed and non-stressed Herceptin
3 days pH 9 stressed originator
Non-stressed originator
1 day pH 9 stressed originator
mAb
mAb with one Lc deamidated
mAb with one Lc deamidated
mAb with both Lc deamidated
mAb with both Lc deamidated
mAb
mAb
23Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
min10 15 20 25 30
mAU
0
100
200
300
400
500
600
Ion exchange chromatographyCEX profile of 1 day pH stressed mAb
2
Reduced CEX peak 1: RPLC profile
Reduced CEX peak 2: RPLC profile
LcHc
1
CEX fraction collection, reduction using DTT and transfer to RPLC method
N→D
Size exclusion chromatography
24Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Proteins
25Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Size exclusion chromatographysu
ppor
t
• 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
SEC analysis of Herceptin highlighting aggregation
mAbmonomer
mAbdimer
Buffer related compound
min0 2 4 6 8 10 12 14 16
mAU
0
10
20
30
40
50
30 cm x 4.6 mm x 3 µm Agilent Bio SEC-3Temp: 24°C Flow: 350 µL/min UV: 214 nmMobile phase: 150 mM phosphate
0.4%
99.6%
26Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
SEC for comparability assessment
min0 2 4 6 8 10 12 14 16
mAU
0
200
400
600
800
min0 2 4 6 8 10 12 14 16
mAU
0
200
400
600
800
mAbmonomer
mAbdimer
Buffer related compound
Originator
Biosimilar
27Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
SEC for comparability assessmentmAb
monomer
mAbdimer
Buffer related compound
min0 2 4 6 8 10 12 14 16
mAU
0
10
20
30
40
50
min0 2 4 6 8 10 12 14 16
mAU
0
10
20
30
40
50
Originator
Biosimilar
Reversed‐phase U/HPLC
28Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Peptides
The power of peptide mapping
• Protein measurement is extremely powerful but does not provide the complete picture nor does it allow to localize modifications
• Which amino acid is glycosylated, oxidized, deamidated, etc.?
• This can be assessed at peptide level following proteolytic digestion with e.g. trypsin
• Peptide measurement is also more powerful towards identity/sequence confirmation
29Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Tryptic peptides Herceptin
Ligh
t Cha
in
B(1
-214
)H
eavy
Cha
in
A(1
-449
)
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 30
Hc
Lc
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW--
--NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
• 62 identity peptides• Modifications• Incomplete and aspecific cleavages• ...
> 100 peptides
Reversed‐phase U/HPLC: Peptide map
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 31
Detailed reversed-phase HPLC peptide map for Herceptin
identity and purity assessment
25 cm x 2.1 mm x 2.7 µm AdvanceBio C18Temp: 60°C Flow: 300 µL/min UV: 214 nmSolv A: 0.05% TFASolv B: 0.05% TFA in ACN 1-45%B, 2-35 min5 µL (2.4 µg)F3C C
O
O-
F3C CO
O-
+H3N
+Lys
S
A
G
F
Y
P
silic
a
C18
• Solvophobic interaction between non‐polar side chains and non‐polar surface• Electrostatic interaction between charged side chains and adsorbed TFA• Elution: increase concentration of organic solvent
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 32
T27
T32
T18
T46
T2
T4T12T20T36T39T48T49
T13
T38T8
T14
T10
T58
T44
T51
T40
T34T23
T62
T11
T19
T24
T30
T56T59 T2
2-23
T22
T5T7
T37-38
T26
T53
T33T25
T57
T29
T47
T21
T45
+ gl
ycan
s
T55
T35
T41
T15T1
T3
T50
T31
T16
T21 pyroGlu
T26
deam
T3 d
eam
T43’
T43
T42
T61
T3’T54T45
T6
Large undigested material*
*
**
T41
ox
* Sample preparation related peaks
Reversed‐phase U/HPLC: Peptide map
LC‐MS based peptide mapping
33Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Compounds extracted out of dataset
• Matching of peptides on sequence (MassHunter BioConfirm)
– Experimental workflow
– In‐silico workflow
LC‐MS based peptide mapping
34Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
protein peptides Experimental MW
Theoretical MWpeptides
Digestion
In-silicodigestion (user defined)
Peptide ID
protein
Mass spectrometry
In-silicomodifications (user defined)
LC‐MS based peptide mapping
35Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Compounds matched onto protein sequence
Compounds extracted out of dataset
LC‐MS based peptide mapping
36Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
• Sequence coverage: 98.8% (655 out of 663 amino acids covered)
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Hc
(A-c
hain
)Lc
(B-c
hain
)
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 37
T45unglycosylated
Originator
T45 glycosylated 99,02 %
T45 unglycosylated 0,98 %
T38
T51T24
T47
T55
T16T6
T37-38
T27
T8
T19
T59
T2
T45 + glycans
Reversed‐phase U/HPLC: Peptide map
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 38
T45 + G0
T45 + G0F
T45 + G1Fa
T45 + G2F
T45 + G1Fb
EEQYNSTYR
• T45 glycopeptide separation
C‐ter C‐ter
N‐terN‐ter
Hc
Lc
C‐ter C‐ter
Reversed‐phase U/HPLC: Peptide map
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 39
T56
T33
T21
T43
T42
T61
T15
T34T23
T25
T3
T3 d
eam
T3’
T26T31
T26
deam
T26
deam
T41T4
1 ox
T18
T13
T14
T62
T22T29
T1T50
T54
T30
T62
+ K
C‐ter C‐ter
N‐terN‐ter
Hc
Lc
C‐ter C‐terM→Mox
N→D
+ K
N→D
Reversed‐phase U/HPLC: Peptide map
Comparability assessment
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 40
Originator
Biosimilar
Comparability assessment
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 41
Originator
Biosimilar
T45 + G0
T45 + G0F
T45 + G1Fa
T45 + G2FT45 + G1Fb
T45 + G0F
T45 + G0T45 + G1F
Undergalactosylation observed in biosimilar
Comparability assessment
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 42
Originator
Biosimilar
ASQDVNTAVAWYQQKPGK Originator Biosimilar
T3 native 91,75 % 86,92 %
T3 deamidation 8,25 % 13,08 %
ASQDVDTAVAWYQQKPGK
ASQDVNTAVAWYQQKPGK
ASQDVDTAVAWYQQKPGK
• Identification of modification sites
The power of mass spectrometry
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 43
Native
Deamidation
Comparability assessment
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 44
Originator
Biosimilar
T62
T62
T62+K--NHYTQKSLSLSPGK
• RPLC x RPLC peptide map of Herceptin
Comprehensive 2D‐LC (LC x LC)
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 4545
Analytical and Bioanalytical Chemistry, issue 1, 2015
• RPLC x RPLC for comparability assessment
Comprehensive 2D‐LC (LC x LC)
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Originator 1 Originator 2 Clone (biosimilar)
4646
• RPLC x RPLC for comparability assessment
Comprehensive 2D‐LC (LC x LC)
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Originator 2
47
T62+K
T62T62
T62+K
MS Originator MS Biosimilar
Hydrophilic interaction chromatography
48Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Glycans
N‐glycan analysis workflow
49Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
2-AB
2-AB
Glycan profile
LC-FLD (MS)2-AB labelingPNGase F
Clean-up
Clean-up2-Aminobenzamide (2-AB)
HILIC: 2‐AB labeled N‐glycans
50Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
• Hydrophilic partitioning between aqueous layer and mobile phase• Elution: increase water concentration
silic
a
Glycan
Glycan
AdvanceBio Glycan Mapping column15 cm x 2.1 mm x 2.7 or 1.8 µm Temp: 55°C Flow: 400 µL/min Fluorescence detectionSolv A: 100 mM NH4-formate pH 4.5Solv B: ACN 80-60%B, 0-38 min
HILIC: 2‐AB labeled N‐glycans
51Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
min8 10 12 14 16 18 20 22 24
LU
-0.5
0
0.5
1
1.5
2
2.5
3
min8 10 12 14 16 18 20 22 24
LU
-0.5
0
0.5
1
1.5
2
2.5
3
1.8 µm fully porous particles
2.7 µm superficiallyporous particles
G1Fa
G1Fb
G2F
G0F
G0
G0-
Glc
NA
c
G0F
-Glc
NA
c
G1-
Glc
NA
cMan5
G1aG1F-GlcNAc
G1b
G0
G0-
Glc
NA
c
G0F
-Glc
NA
c
G1-
Glc
NA
c
G0F
Man5G1a
G1F-GlcNAc
G1Fa
G1Fb
G2F
The power of mass spectrometry
52Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
AB
AB
AB
ABAB
ABAB
AB
AB
AB
AB
AB
AB
AB
AB
• MS/MS spectrum of 2AB‐labeled G0F
min10 15 20 25 30
LU
0
2.5
5
7.5
10
12.5
15
17.5
min10 15 20 25 30
LU
02468
10121416
Comparability assessment
53Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Originator
Biosimilar
Undergalactosylation observed in biosimilar
G0
G0F
G1Fa
G1FbG2F
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
min10 15 20 25 30
LU
0
2
4
6
8
min10 15 20 25 30
LU
02468
10
min10 15 20 25 30
LU
02.5
57.510
12.515
min10 15 20 25 30
LU
02468
1012
min10 15 20 25 30
LU
02468
1012
54
Biosimilar
Biosimilar ‐ Cell culture optimization• Bringing glycosylation within originator specifications by adding
uridine, galactose and manganese to the CHO growth medium
G0
G0F
G1F G2F
Biosimilar: 4x
Biosimilar: 24x
Biosimilar: 8x
Biosimilar: 16x
55Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar
Biosimilar ‐ Cell culture optimization
1.8 µm fully porous HILIC particles
GlycanRelative intensity
Biosimilar Biosimilar4x
Biosimilar8x
Biosimilar16x
Biosimilar24x
Specificationsoriginators
% G0‐GlcNAC 0.55 0.49 0.47 0.53 0.61 0.77 +/‐ 0.09
% G0F‐GlcNAc 5.35 2.72 2.15 1.78 1.67 1.88 +/‐ 0.87
% G0 1.57 2.67 1.98 2.35 2.02 4.35 +/‐ 0.29
% G1‐GlcNAc 0.39 0.32 0.35 0.45 0.51 1.07 +/‐ 0.34
% 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 1.89 +/‐ 0.35
% G1a + % G1F‐GlcNAc 0.55 1.35 1.34 1.51 1.44 2.61 +/‐ 0.30
% G1b 0.17 0.32 0.29 0.37 0.38 0.69 +/‐ 0.20
% G1Fa 7.76 22.66 25.58 25.83 25.91 27.30 +/‐ 3.48
% G1Fb 3.62 8.11 8.70 8.75 8.62 9.13 +/‐ 0.89
% G1F 11.38 30.77 34.28 34.57 34.53 36.44 +/‐ 4.38
% G2F 1.01 6.66 7.80 8.03 7.98 5.92 +/‐ 2.47
• Bringing glycosylation within originator specifications by adding uridine, galactose and manganese to the CHO growth medium
Acknowledgement
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 56
• Isabel Vandenheede, Emmie Dumont and Pat Sandra (RIC, Kortrijk, Belgium)
• The colleagues from the biopharmaceutical industry who trigger and inspire us in developing and applying chromatographic and mass spectrometric methodologies and strategies to tackle their challenging requests
• Maureen Joseph, Gina Goggings, Linda Lloyd, Phu Duong (Agilent Technologies, Wilmington, Delaware)
www.richrom.com
Strategies for the Separation and Characterization of Protein Biopharmaceuticals
Improved LC Column Choices for Bioseparations
January 28, 2015
February 12, 2015
Confidentiality Label
1
Strategies of Column Selection for Separation and Characterization of Protein Biopharmaceuticals
February 12, 2015
Confidentiality Label
2
Issue Challenge Reason SolutionPeak tailing
• Lowerresolution
• Less accuracy
• Secondary ionicinteractions
• High number of pos charges on proteins
• Stationary phase with limitedaccess to residual silanols
• Ion-pairing reagent• Higher temperature
Peak broadening
• Lower resolution
• Reduced sensitivity
• Low Dm of large molecules
• Limited access topores
• Widepore phases• Higher temperature• Efficient stationary phase
(sub 2 µm, superficially porous)
Adsorption • Poor recovery• Less sensitivity
• Hydrophobicity • Less hydrophobic stationaryphases
• Stronger solvent• High temperatures
Column parameters are important to solve problems of efficiency/resolution and recovery for improved LC and LC/MS characterization of proteins.
Reversed-phase LC Issues and Solutions
Reversed Phase Column Choices Improve Efficiency/Resolution and RecoveryAdvanceBio RP-mABThe optimum high speed, large molecule resolution for use with both HPLC and UHPLC systems• Superficially porous, 3.5um particle
The most popular phases for proteins, plus a unique selectivity.• C4, C8, Diphenyl
ZORBAX RRHD 300Å, 1.8umFast, high resolution UHPLC analysis of proteins, including intact mAbs, and protein fragments• Totally porous 1.8um silica particles with
300Å pores• 1200 bar for UHPLC use• Suitable for intact, fragments and digests
The most popular phases for proteins plus a unique selectivity.• SB-C3, SB-C8, SB-C18, Diphenyl• StableBond bonding for long lifetime with
TFA ion-pairing reagent
February 12, 2015
Confidentiality Label
3
3.5 um3.0 um
0.25 um
450Å pores
Superficially porous particle
Fast Intact mAb Analysis and Comparison of PhasesShort 3 minute separation, with each phase unique.
Method Parameters Column dimensions: 2.1 x 100 mmMobile 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
Gradient: 10-58% B in 4 min, 1 min wash at 95% B, 1 min re-equilibration at 10% BSample: 5 μL injection of Humanized Recombinant Herceptin Variant IgG1 Intact from Creative Biolabs (1 mg/mL) Temperature: 80 °CDetection: UV @ 254nm
February 12, 2015
4
min1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5
mAU
0
20
40
60
80
100
120
140
DAD1 H, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-21 08-02-26\1443508-52-0006.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-20 09-06-44\1435601-25-0037.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-19 15-36-09\DIP143501-3-047.D)
min1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5
mAU
-4
-2
0
2
4
6
8
10
12
14
DAD1 H, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-21 08-02-26\1443508-52-0006.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-20 09-06-44\1435601-25-0037.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-19 15-36-09\DIP143501-3-047.D)
AdvanceBio RP-mAb C4AdvanceBio RP-mAb SB-C8AdvanceBio RP-mAb Diphenyl
Agilent Technologies
2 2.5
Reversed Phase Peptide Mapping – Resolution Maximized with SPP AdvanceBio Peptide MappingUHPLC column efficiency and resolution for your complete peptide map • HPLC and UHPLC• 2.7um superficially porous particle• LC and LC/MS• Minimal peak tailing• Optimal C18 bonding
February 12, 2015
Confidentiality Label
5
8x10
0
0.75
1.75
2.75
3.75
Acquisition Time (min)1 2 3 4 5 6 7 8 9 101112131415161718192021222324252627282930313233343536373839
40 min. Analysis0.2mL/min140 Bar
8x10
0
0.6
1.6
2.6
3.6
4.6
Acquisition Time (min)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.56 6.5 7 7.5 8 8.5 9 9.510 10.511 11.512 12.513
14 min. Analysis0.6mL/min433 Bar
TIC2.1 x 100mm, 2.7um
2.7um1.7um
0.50 um
120Å pores
Choices for Other Modes of Chromatography - SEC
SEC – for aggregation analysis• Columns are porous silica with typical
lengths of 250 or 300 mm• Typical flow rate is 1.0 mL/min on a
7.8 mm ID column or 0.35 mL/min on a 4.6 mm ID column
• To increase resolution (through increased pore volume) run columns in series (costs time!)
• To increase resolution, use smaller particle sizes (3um)
• To reduce secondary interactions and improve recovery maximize inertness
BioSEC-3, 7.8 x 300mm, 3um• 3 m particle• Proprietary hydrophilic coating to
prevent secondary interaction• 100Å, 150Å, 300Å pore sizes• High efficiency and resolution• Faster SEC separations• Can be run with low salt buffers
February 12, 2015
Confidentiality Label
6
Improved SEC Efficiency With Smaller Particles
Column: Bio SEC-3 300Å and Bio SEC-5 300ÅBuffer: 150 mM Phosphate buffer, pH 7Flow rate: 1.0 mL/min for 7.8 x 300 mm Temperature: Ambient (~23 °C)Detection: UV 214nmInjection: 10 µL (3 L for 4.6 x 300 mm)Sample: 1) Thyroglobulin (1.0 mg/mL), 670 kD; 2) BSA dimer, 132 kD; 3) BSA (1.0 mg/mL), 66 kD; 4) Ribonuclease A (1.0 mg/mL), 13.7 kD, and 5) Uracil (2.5 g/mL), 120 D.
Min0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Agilent Bio SEC-3, 300Å,7.8 x 300 mm93 bar
Agilent Bio SEC-5, 300Å,7.8 x 300 mm45 bar
Peak Protein Efficiency Gain
SEC-3, 300Å (7.8x300mm)
SEC-5, 300Å (7.8x300mm)
1 Thyroglobulin 2.2X 2460 1120
2 BSA Dimer 1.9X 5100 2720
3 BSA 2.0X 13090 6590
4 Ribonuclease A 2.0X 22000 11160
5 Uracil 1.4X 38500 27860
Choices for Other Modes of Chromatography - IEX
IEX – for charge variants• Typically non-porous 10um
polymeric particles, 250mm long• Separation with high salt or pH
changes• Non-specific interactions with
surfaces can reduce resolution• Metal ions eluting from instrument
can lower resolution and cause column poisoning
• To increase resolution choose smaller particle sizes (5 and 3um)
• To maximize recovery choose inert systems
BioMAb 3 or 5um 4.6 mm• Non-porous PS/DVB particles• Uniform polymeric hydrophilic coating
and WCX layer, specifically designed for antibody separations with minimal non-specific interactions
• Fully Bio-inert choices for maximum recovery - 10 µm, 5 µm with PEEK
• Also available in 3 µm and 1.7 µm particle sizes for highest resolution
February 12, 2015
Confidentiality Label
8
Improving Ion Exchange Chromatography (IEX)
Better peak shape and higher efficiency were achieved with a smaller particle size on the Agilent Bio MAb 5 vs. Bio MAb 10 column.
9Characterization of mAb
2/12/2015
min0 5 10 15 20 25 30 35
mAU
0
100
200
300
400
500
1.4
31
1.5
59
2.7
25
24.8
28
26.5
60
33.9
02
05-30% B in 30min
Incomplete separation due to protein/ system interactions. Massive fronting and tailing makes quantitation impossible.
Resolve charge variants with BioInert LC and column.
Choices for Other Modes of Chromatography – HILICReleased glycan analysis by HPLC-FLD
1260 Infinity Bio-inert HPLC/FLD
AdvanceBioGlycan Mapping,
2.7 um
AdvanceBioGlycan Mapping,
2.7 um+
AdvanceBioGlycan Mapping,
1.8 um
AdvanceBioGlycan Mapping,
1.8 um+
1290 Infinity UHPLC
(superficially porous)
10
(totally porous)
Why Amide HILIC?Selectivity is stable over column lifetimeHigh peak capacityRT increases with size, depending on monosaccharide type and position
Columns can Reduce Challenges in Chromatography for the Characterization of Monoclonal Antibodies
Titer determination and purification Affinity Chromatography
Protein identification and impurity profilingReversed-phase chromatography (RP)
Glycan analysisHydrophilic interaction chromatography (HILIC)
Charge variant analysisIon exchange chromatography (IEX)
Aggregation analysisSize exclusion chromatography (SEC)
February 12, 2015
11
Agilent Technologies