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Strategies for the Separation and
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)
20% of the total pharmaceutical market

Within the current decade, more than 50% of new drug
approvals will be biologics
Strategies 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
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
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
EVQLVESGGGLVQPGGSLRLSCAAS
GFNIKDTYIHWVRQAPGKGLEW--
N-ter
N-ter
Hc
Lc
C-ter
C-ter
--NYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Typical characteristics
Amino acid sequence

Structural integrity
Higher order structures
Aggregation
S-S bridges
N- and O-glycosylation
N- and C-terminal sequence
Charge variants
Deamidation/isomerization
Oxidation
Clipping
N-ter
N-ter
Hc
Lc
C-ter
C-ter
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Characteristics determined at different levels
Protein
Acid hydrolysis
Amino acids
Trypsin
digestion
MW, structural integrity, charge

variants, aggregation, modifications
PNGase F
Peptides
Amino acid sequence, modifications,

modification sites, disulfide bridges, etc.
Sugars
N-glycans
A wide range of separation modes
Charge
Size
Hydro(phob/phil)icity
Affinity
CEX, AEX
SEC
RPLC, HIC, HILIC
Affinity Chromatography
Reversed-phase U/HPLC
Proteins
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
RPLC analysis for identity and purity determination of Herceptin
Hc
Lc
Hc
Fab
Lc
Fab
Fc
Intact
Fc
10 cm x 2.1 mm x 1.8 m Zorbax 300 SB-C8

Temp: 80C Flow: 200 L/min
UV: 214 nm
Solv A: 0.1% TFA
Solv B: 0.1% TFA in ACN
30-38.6%B, 2-25 min
11
Reversed-phase U/HPLC Mass

Spectrometry
RPLC-UV-MS of Herceptin Lc and Hc
Deconvoluted spectra
12
Reversed-phase U/HPLC for

comparability assessment
Lc
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

x10
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

G0F + C-terminal Lys
x10 4
50724.3
2.5
2
1.5
1
C-ter
--NHYTQKSLSLSPGK
0.5
0
G0F
x10 5
3
50596.7
Undergalactosylation observed in biosimilar
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
Widepore Poroshell for comparability

assessment
x10 3
1
Advance Bio RP-mAb

5 cm x 4.6 mm x 3.5 m 450 C4
Temp: 80C Flow: 1 mL/min UV: 214 nm
Solv A: 0.1% TFA
Solv B: 0.1% TFA in 90% ACN
30-42.5%B, 0-6 min
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
Response Units vs. Acquisition Time (min)
Remicade
biosimilar
16

assessment
x10 3
1
Advance Bio RP-mAb

5 cm x 4.6 mm x 3.5 m 450 C4
Solv A: 0.1% TFA
30-42.5%B, 0-6 min
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
Differences in hydrophobicity due

to a 2-point mutation in the AA
sequence of the biosimilar
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
Widepore Poroshell zero carry-over

Remicade
(4 g o.c.)
Advance Bio RP-mAb

5 cm x 4.6 mm x 3.5 m 450 C4
Solv A: 0.1% TFA
30-42.5%B, 0-6 min
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
Blank
18

assessment
Remicade
Remicade
biosimilar
Hc
Lc
Advance Bio RP-mAb

5 cm x 4.6 mm x 3.5 m 450 C4
Solv A: 0.1% TFA
30-42.5%B, 0-6 min
Differences in hydrophobicity due to a 2-point

mutation in the AA sequence of the biosimilar
compared
19
Ion exchange
chromatography
Proteins
20
Ion exchange chromatography

WCX analysis (n=5) of Herceptin highlighting charge variants
_
_
_
_
+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
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

mAb
mAU
600
WCX analysis of stressed

and non-stressed Herceptin
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

2
mAU
600
CEX profile of 1 day pH

stressed mAb
500
400
CEX fraction collection,

reduction using DTT and
transfer to RPLC method
300
200
100
0
10
15
20
25
30
min
Reduced CEX peak 1: RPLC profile
ND
Reduced CEX peak 2: RPLC profile

Hc
Lc
23
Size exclusion
chromatography
Proteins
24
Size exclusion chromatography

30 cm x 4.6 mm x 3 m Agilent Bio SEC-3
UV: 214 nm
Mobile phase: 150 mM phosphate
support
SEC analysis of Herceptin

highlighting aggregation
mAb
monomer
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
SEC for comparability assessment

mAb
monomer
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
SEC for comparability assessment

mAb
monomer
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
Peptides
28
The power of peptide mapping

Protein measurement is extremely powerful but does
not provide the complete picture nor does it allow
localized 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
29
Tryptic peptides Herceptin
Heavy Chain
A(1-449)
Light Chain
B(1-214)
Hc
Lc
QQGNVFSCSVMHEALHNHYTQKSLSLSPG
62 identity peptides
Modifications
Incomplete and aspecific cleavages
...
> 100 peptides
30
Reversed-phase U/HPLC: Peptide map

Detailed reversed-phase HPLC
peptide map for Herceptin
identity and purity assessment
C18
O
F3 C C
O-
25 cm x 2.1 mm x 2.7 m AdvanceBio C18

Temp: 60C Flow: 300 L/min UV: 214 nm
Solv A: 0.05% TFA
Solv B: 0.05% TFA in ACN
1-45%B, 2-35 min
5 L (2.4 g)
+H N
3
silica
A
G
F
Y
O
F3 C C
O-
P
+Lys
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
31

*
* *
Large undigested material
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
* Sample preparation related peaks

32
LC-MS based peptide mapping

Compounds extracted out
of dataset
33

Matching of peptides on sequence (MassHunter BioConfirm)
Experimental workflow
protein
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

Compounds extracted out
of dataset
Compounds matched onto

protein sequence
35
Hc (A-chain)
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKG
RFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP
IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
Lc (B-chain)
Sequence coverage: 98.8% (655 out of 663 amino acids covered)
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSG
TDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN
FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
TKSFNRGEC
36

Originator
T45 glycosylated
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

T45 glycopeptide separation
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
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
T45 + G0F
Originator
T45 + G1Fa
T45 + G1Fb
T45 + G2F
T45 + G0
T45 + G0F
Biosimilar
Undergalactosylation
observed in biosimilar
T45 + G1F
T45 + G0
41
ASQDVNTAVAWYQQKPGK
Originator
Biosimilar
T3 native
91,75 %
86,92 %
T3 deamidation
8,25 %
13,08 %
Originator
ASQDVDTAVAWYQQKPGK
ASQDVNTAVAWYQQKPGK
Biosimilar
ASQDVDTAVAWYQQKPGK
42
The power of mass spectrometry

Identification of modification sites
Native
Deamidation
43
Originator
T62
Biosimilar
T62
--NHYTQKSLSLSPGK
T62+K
44
Comprehensive 2D-LC (LC x LC)

RPLC x RPLC peptide map of Herceptin
Analytical and Bioanalytical Chemistry, issue 1, 2015
45

RPLC x RPLC for comparability assessment
Originator 1
Originator 2
Clone (biosimilar)
46

RPLC x RPLC for comparability assessment
Originator 2
MS Originator
T62
T62
T62+K
MS Biosimilar
T62+K
47
Hydrophilic interaction
chromatography
Glycans
48
N-glycan analysis workflow
PNGase F
2-AB labeling
LC-FLD (MS)
2-AB
Glycan profile
2-AB
Clean-up
Clean-up
2-Aminobenzamide (2-AB)
49
HILIC: 2-AB labeled N-glycans

AdvanceBio Glycan Mapping column
15 cm x 2.1 mm x 2.7 or 1.8 m
Fluorescence detection
Solv A: 100 mM NH4-formate pH 4.5
Solv B: ACN
80-60%B, 0-38 min
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
HILIC: 2-AB labeled N-glycans

G0F
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
The power of mass spectrometry

MS/MS spectrum of 2AB-labeled G0F
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
52
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
Undergalactosylation observed in biosimilar
53
Biosimilar - Cell culture optimization

Bringing glycosylation within originator specifications by adding
uridine, galactose and manganese to the CHO growth medium
G0F
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 - Cell culture optimization

Bringing glycosylation within originator specifications by adding
uridine, galactose and manganese to the CHO growth medium
1.8 m fully porous HILIC particles
Relative intensity
Glycan
Biosimilar
Biosimilar
4x
Biosimilar
8x
Biosimilar
16x
Biosimilar
24x
Specifications
originators
% 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
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
Strategies for the

Separation and
Characterization of
Protein
Biopharmaceuticals
Improved LC Column
Choices for Bioseparations
January 28, 2015
Confidentiality Label
January 28, 2015
57
Strategies of Column Selection for Separation and

Characterization of Protein Biopharmaceuticals
Reversed-phase LC Issues and Solutions
Issue
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
Less hydrophobic stationary

phases
Stronger solvent
High temperatures
Peak
broadening
Adsorption
Stationary phase with limited

access to residual silanols
Ion-pairing reagent
Higher temperature
Column parameters are important to solve problems of efficiency/resolution and recovery for
improved LC and LC/MS characterization of proteins.
January 28, 2015
58
Reversed Phase Column Choices Improve

Efficiency/Resolution and Recovery
AdvanceBio RP-mAB
ZORBAX RRHD 300, 1.8um
The optimum high speed, large

molecule resolution for use with both
HPLC and UHPLC systems
Fast, high resolution UHPLC analysis of

proteins, including intact mAbs, and
protein fragments
Superficially porous, 3.5um particle
Totally porous 1.8um silica particles with

300 pores
0.25 um
1200 bar for UHPLC use

3.0 um
450 pores
3.5 um
Superficially
porous
particle
Suitable for intact, fragments and digests
The most popular phases for proteins

plus a unique selectivity.
SB-C3, SB-C8, SB-C18, Diphenyl
The most popular phases for proteins,

plus a unique selectivity.
StableBond bonding for long lifetime with

TFA ion-pairing reagent
C4, C8, Diphenyl

January 28, 2015
59
Fast Intact mAb Analysis and Comparison of Phases

Short 3 minute separation, with each phase unique.
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)
mAU
AdvanceBio RP-mAb C4
AdvanceBio RP-mAb SB-C8
AdvanceBio RP-mAb Diphenyl
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)
mAU
14
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
Gradient: 10-58% B in 4 min, 1 min wash at 95% B, 1 min re-equilibration at 10% B

Sample: 5 L injection of Humanized Recombinant Herceptin Variant IgG1 Intact from Creative Biolabs (1
mg/mL)
Temperature: 80 C
Detection: UV @ 254nm
Agilent Technologies
January 28, 2015
60
Reversed Phase Peptide Mapping Resolution

Maximized with SPP
x108
3.75
AdvanceBio Peptide Mapping

2.75
UHPLC column efficiency and resolution

for your complete peptide map
40 min.
Analysis
0.2mL/min
140 Bar
TIC
2.1 x 100mm, 2.7um
1.75
HPLC and UHPLC

2.7um superficially porous particle
0.75
LC and LC/MS
0
1 2 3 4 5 6 7 8 9 101112131415161718192021222324252627282930313233343536373839
Minimal peak tailing
Acquisition Time (min)
Optimal C18 bonding
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
Acquisition Time (min)
January 28, 2015
61
Choices for Other Modes of Chromatography - SEC

SEC for aggregation analysis
BioSEC-3, 7.8 x 300mm, 3um
Columns are porous silica with typical

lengths of 250 or 300 mm
3 m particle
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!)
Proprietary hydrophilic coating to

prevent secondary interaction
100, 150, 300 pore sizes
High efficiency and resolution
Faster SEC separations

Can be run with low salt buffers
To increase resolution, use smaller

particle sizes (3um)
To reduce secondary interactions and
improve recovery maximize inertness
January 28, 2015
62
Improved SEC Efficiency With Smaller Particles
Agilent Bio SEC-3, 300,

7.8 x 300 mm
93 bar
Protein
Efficiency
Gain
SEC-3, 300 SEC-5, 300

(7.8x300mm) (7.8x300mm)
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
Column: Bio SEC-3 300 and Bio SEC-5 300

Buffer: 150 mM Phosphate buffer, pH 7
Flow rate: 1.0 mL/min for 7.8 x 300 mm
Temperature: Ambient (~23 C)
Detection: UV 214nm
Injection: 10 L (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)
Agilent Bio SEC-5, 300,

7.8 x 300 mm
45 bar
1
Peak
8
Min
10 11 12 13 14 15
Ribonuclease A (1.0 mg/mL), 13.7 kD, and 5) Uracil

(2.5 g/mL), 120 D.
Choices for Other Modes of Chromatography - IEX

IEX for charge variants
BioMAb 3 or 5um 4.6 mm
Non-porous PS/DVB particles
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
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 m, 5 m with PEEK
Also available in 3 m and 1.7 m
particle sizes for highest resolution
January 28, 2015
64
Improving Ion Exchange Chromatography (IEX)
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
Resolve charge variants with BioInert LC

0 column.
and
Better peak shape and higher efficiency

were achieved with a smaller particle
size on the Agilent Bio MAb 5 vs. Bio
MAb 10 column.
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
Incomplete separation due to protein/

system interactions. Massive fronting and
tailing makes quantitation impossible.
Characterization of mAb
65
1/28/2015
Choices for Other Modes of Chromatography HILIC

Released glycan analysis by HPLC-FLD
AdvanceBio
Glycan Mapping,
2.7 um
(superficially porous)
1260 Infinity
Bio-inert HPLC/FLD
Why Amide HILIC?

Selectivity is stable over column lifetime
High peak capacity
RT increases with size, depending on
monosaccharide type and position
+
1290 Infinity UHPLC
AdvanceBio
Glycan Mapping,
1.8 um
(totally porous)
66
Columns can Reduce Challenges in Chromatography

for the Characterization of Monoclonal Antibodies
Titer determination and purification
Affinity Chromatography
Protein identification and impurity profiling
Reversed-phase chromatography (RP)
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

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