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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
Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

Protein characterization
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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

Protein characterization
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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

Protein characterization
Characteristics determined at different levels
Protein
Acid hydrolysis

Amino acids

Amino acid composition

Trypsin
digestion

MW, structural integrity, charge


variants, aggregation, modifications
PNGase F

Peptides

Amino acid sequence, modifications,


modification sites, disulfide bridges, etc.

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

Sugars

N-glycans

A wide range of separation modes

Charge
Size
Hydro(phob/phil)icity
Affinity

CEX, AEX
SEC
RPLC, HIC, HILIC
Affinity Chromatography

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

Reversed-phase U/HPLC
Proteins

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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Reversed-phase U/HPLC
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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

11

Reversed-phase U/HPLC Mass


Spectrometry
RPLC-UV-MS of Herceptin Lc and Hc

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

20
min

13

Reversed-phase U/HPLC for


comparability assessment
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)

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

14

Reversed-phase U/HPLC for


comparability assessment
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)

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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)

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

Remicade
biosimilar

16

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

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

Response Units vs. Acquisition Time (min)

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

Response Units vs. Acquisition Time (min)

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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
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 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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

18

Widepore Poroshell for comparability


assessment
Remicade
Remicade
biosimilar

Hc
Lc

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

Differences in hydrophobicity due to a 2-point


mutation in the AA sequence of the biosimilar
compared

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

19

Ion exchange
chromatography
Proteins

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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
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)

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

30

35

min

pI high
(BASIC)

21

Ion exchange chromatography


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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

30

min

22

Ion exchange chromatography


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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

23

Size exclusion
chromatography
Proteins

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

24

Size exclusion chromatography


30 cm x 4.6 mm x 3 m Agilent Bio SEC-3
Temp: 24C Flow: 350 L/min
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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

14

16

min

27

Reversed-phase U/HPLC
Peptides

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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Tryptic peptides Herceptin

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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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Reversed-phase U/HPLC: Peptide map


*

* *

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


Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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LC-MS based peptide mapping


Compounds extracted out
of dataset

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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LC-MS based peptide mapping


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)

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

34

LC-MS based peptide mapping


Compounds extracted out
of dataset

Compounds matched onto


protein sequence

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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LC-MS based peptide mapping

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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

36

Reversed-phase U/HPLC: Peptide map


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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

T45
unglycosylated

37

Reversed-phase U/HPLC: Peptide map


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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

38

Reversed-phase U/HPLC: Peptide map


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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

T30
T61
T56

39

Comparability assessment
Originator

Biosimilar

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

41

Comparability assessment
ASQDVNTAVAWYQQKPGK

Originator

Biosimilar

T3 native

91,75 %

86,92 %

T3 deamidation

8,25 %

13,08 %

Originator

ASQDVDTAVAWYQQKPGK

ASQDVNTAVAWYQQKPGK

Biosimilar

ASQDVDTAVAWYQQKPGK

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

42

The power of mass spectrometry


Identification of modification sites
Native

Deamidation

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

43

Comparability assessment
Originator

T62

Biosimilar

T62
--NHYTQKSLSLSPGK

T62+K

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

44

Comprehensive 2D-LC (LC x LC)


RPLC x RPLC peptide map of Herceptin

Analytical and Bioanalytical Chemistry, issue 1, 2015

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

45

Comprehensive 2D-LC (LC x LC)


RPLC x RPLC for comparability assessment
Originator 1

Originator 2

Clone (biosimilar)

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

46

Comprehensive 2D-LC (LC x LC)


RPLC x RPLC for comparability assessment
Originator 2

MS Originator

T62

T62

T62+K

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

MS Biosimilar

T62+K

47

Hydrophilic interaction
chromatography
Glycans

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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)

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

49

HILIC: 2-AB labeled N-glycans


AdvanceBio Glycan Mapping column
15 cm x 2.1 mm x 2.7 or 1.8 m
Temp: 55C Flow: 400 L/min
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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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

Undergalactosylation observed in biosimilar

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

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.
Confidentiality Label
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


Confidentiality Label
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)

Confidentiality Label
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

Confidentiality Label
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

Confidentiality Label
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