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Shanhua Lin, Julia Baek, Mark Tracy, and Chris PohlThermo Fisher Scientific, Sunnyvale, USA

Universal Ion Exchange Chromatography Separation Platform

For research use only. Not for use in diagnostic procedures.

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Biopharama and Pharma Landscape: Top 15 Best Selling Drugs of 2018 Ranking Drug Maker 2017 sales 2018 sales Growth

1 Humira®(adalimumab) AbbVie $19.936 billion $19.936 billion 8.2%

2 Eliquis® (apixaban) BMS and Pfizer $7.395 billion $9.872 billion 33.5%

3 Revlimid (lenalidomide) Celgene $8.187 billion $9.685 billion 18.3%

4 Opdivo® (nivolumab) BMS $5.763 billion $7.570 billion 31.4%

5 Keytruda® (pembrolizumab) Merck $3.809 billion $7.171 billion 88.3%

6 Enbrel® (etanercept) Amgen and Pfizer $7.885 billion $7.126 billion –9.6%

7 Herceptin® (trastuzumab) Roche (Genentech) $7.013 billion $6.981 billion –0.5%

8 Avastin® (bevacizumab) Roche (Genentech) $6.686 billion $6.847 billion 2.4%

9 Rituxan® (rituximab) Roche (Genentech) $7.298 billion $6.750 billion –7.5%

10 Xarelto® (rivaroxaban) Bayer and JNJ $6.234 billion $6.589 billion 5.8%

11 Eylea® (aflibercept) Regeneron $5.830 billion $6.551 billion 12.4%

12 Remicade® (infliximab) JNJ $7.152 billion $5.908 billion –17.4%

13 Prevnar 13 (Pneumococcal 13-valent Conjugate Vaccine

Pfizer $5.601 billion $5.802 billion 3.6%

14 Stelara (ustekinumab) Janssen Biotech $4.011 billion $5.156 billion 28.5%

15 Lyrica® (pregabaliln) Pfizer $5.065 billion $4.970 billion –1.9%

https://www.genengnews.com/a-lists/top-15-best-selling-drugs-of-2018/

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• Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products• Purity: Due to the unique biosynthetic production process and molecular characteristics of biotechnological

and biological products, the drug substance can include several molecular entities or variants. When these molecular entities are derived from anticipated post-translational modification, they are part of the desired product. When variants of the desired product are formed during the manufacturing process and/or storage and have properties comparable to the desired product, they are considered product-related substances and not impurities (section II.A.1), e.g. charge variants due to deamidation.

Analytical Tests for Recombinant Therapeutics

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• mAb charge variant analysis (Cation exchanger)

• Acidic: Sialylation, deamidation, C-terminal lysine cleavage

• Basic: N-terminal cyclization of glutamic acid, methionine oxidation

IEC is a Common Method for Protein Charge Variant Analysis

• Acidic protein charge variant analysis (Anion exchanger)

• Acidic: Sialylation, deamidation, C-terminal lysine cleavage

• Basic: N-terminal cyclization of glutamic acid, methionine oxidation

0.0 26.00

95

Time (min)

Abso

rban

ce (m

AU)

acidic basic

main

NivolomabProPac Elite WCXSalt gradientpH 6.5

mAb144 kDapI 6.1-8.5

0.0 5.0 10.0 15.0 20.0 25.0-5.0

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

min

acidicbasic

Coagulation factorProPac SAX-10Salt gradientpH 8.5

Fusion protein120 kDapI 5-6

main

5

• Interactions of protein cationic sites with anionic sites of stationary phase determine separation

• Chromatographic parameters influence separation

Two Types of Ion-Exchange Methods

Infliximab

mAU Acidic

+0 Lysine

+1 Lysine

+2 Lysine

-10.0

50.0

70.0

Time, min0.0 5.0 10.0 15.0

-5.0

20.0

40.0

50.0

0.0 5.0 10.0 15.0Time, minutes

mAU Acidic

+0 Lysine

+1 Lysine

+2 Lysine

Infliximab

Salt gradient pH gradient

• Mobile phase pH• Salt gradient slope

• Electrolyte concentration• pH gradient slope

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Salt Gradient Method and pH Gradient Method

++

+- ---

-----

-

-- --

Positive

Salt gradient method pH gradient method

++

+- -

--

-----

-

-- --Na+

Cl-

Na+

Cl-

Neutral

--

Negative

pH

Time (min)

[Sal

t]

Time (min)

7

Two Methods for Protein/mAb Separation on IEX Columns

• Weaken ionic interactions between analyte and stationary phase by increasing the salt concentration

• Salt disrupts protein-solid phase interactions

• Most widely used method

• Relatively simple to make the buffer

• Buffer design (pH, salt concentration) can optimize the separation

Salt gradient

• Change the pH of the eluent at constant ionic strength

• The change in protein charge disrupts protein-solid phase interactions

• Possible to predict elution profile with pI value

• Lower salt concentration in collected fractions

• Thermo Scientific™ CX-1 pH Gradient Buffers provide a linear pH gradient

pH gradient

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Non-linear pH Gradient Method

• Piperazine , pKa 5.33, 9.73 (6 mM): Cation (Binds to stationary phase), bivalent• Imidazole, pKa 6.95 (11 mM): Hazardous material• Tris, pKa 8.1 (9.6 mM): Cation (Binds to stationary phase)

No-column

Thermo Scientific™ ProPac™ WCX-10

Thermo Scientific™ MAbPac™ SCX-10

Rea, J.C., et al. Validation of a pH gradient-based ion-exchange chromatography Method for high-resolution monoclonal antibody charge variant separations, J. Pharm. Biomed. Anal. 2011, Jan 25;54(2), 317-23.

0.0 5.0 10.0 15.06.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

11.5

Time (min)20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0

pH

9

MES MOPS TAPS CAPSO6.1 7.2 8.4 9.6

Buffer Development StrategyBuffer Development Strategy

• Replace cationic buffer components with zwitterionic buffer species (Good’s Buffers)

• These buffer species contain one amine group and one sulfonic acid group. They do not bind to the stationary phase in the pH range of 6-10.

• They are not repelled by the stationary phase so they can buffer the stationary phase.

pKa

10

MES-MOPS-TAPS-CAPSO Buffer Cocktail

y = 0.1577x + 4.9755R² = 0.9996

5.5

6.5

7.5

8.5

9.5

10.5

0 10 20 30 40

Mea

sure

d pH

val

ue

Retention Time [min]

Measured ValueLinear (Measured Value)

US8921113 B2: Buffer kit and method of generating a linear pH gradient

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Effect of Buffer Salt Repelled or Adsorbed to the Stationary phase

5.5

6

6.5

7

7.5

8

8.5

9

9.5

10

10.5

0 5 10 15 20 25 30 35 40

Mea

sure

d pH

Val

ue

Time (min)

Comparison of Three Buffer Formulation

MES-MOPS-TAPS-CAPSOMES-Phosphate-TAPS-CAPSOMES-MOP-Tris-CAPSO

TAPS Tris

MOPS Phosphate

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Thermo Scientific™ CX-1 pH Gradient Buffer

Buffer A Buffer B

pH 5.6 10.2

Form Liquid Liquid

Concentrate 10X 10X

Shipping Room temp. Room temp.

Storage temp. 4 ~ 8 °C 4 ~ 8 °C

pH gradient applications

Dilute buffers 10-fold with DI water A linear pH gradient (pH 5.6 – 10.2

is generated by running a linear pump gradient from 100% Buffer A to 100% Buffer B

Platform, fast, and high-resolution!

Question: In addition to pH gradient applications, could we perform both CEX and AEX with these buffers, running salt gradient at different pHs?

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Universal IEC Platform Set Up: Chromatography Conditions

• Column

• ProPac WCX-10 for cation exchange• ProPac SAX-10 for anion exchange

• Format: 4x150 mm

• Flow rate: 1 mL/min

• Temperature: 30 ºC

• Salt gradient: 0 to 200 mM NaCl (0 to 20%B) gradient in 15 minutes for ProPac WCX-10

• pH gradient: 10%C, 0%D (pH 5.6) → 0%C, 10%D (pH 10.2) in 15 minutes

• Salt gradient: 20 to 500 mM NaCl (2% to 50%B) gradient in 20 minutes for ProPacSAX-10

DI water 1 M NaCl

10X

MESMOPSTAPS

CAPSO

pH 5.6

10X

MESMOPSTAPS

CAPSO

pH 10.2

A B C D

Add up to 10%Add up to 90%

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Program No.

% Eluent C % Eluent D Nominal pH

I 10 0 5.60

II 9 1 6.06

III 8 2 6.52

IV 7 3 6.98

V 6 4 7.44

VI 5 5 7.9

VII 4 6 8.36

VIII 3 7 8.82

IX 2 8 9.28

X 1 9 9.74

XI 0 10 10.2

Method Scouting

ProgramNo. % Eluent A % Eluent B [NaCl] (mM)

I 89 1 10

II 87 3 30

III 84 6 60

V 81 9 90

Salt gradient pH gradient

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Regulating the Mobile Phase pH by Proportioning Eluents C and D

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 56.05.00

10.00

10.50

CX-1 pH Grad B: 0.0 %1.0

2.03.0

4.05.0

6.07.0

8.09.0

10.0

CX-1 pH Grad A: 10.0 %9.0

8.07.0

6.05.0

4.03.0

2.01.0

0.0

pH

5.53

6.01

6.49

6.98

7.50

8.05

8.53

9.01

9.44

9.8510.22

Eluent CEluent D

pH trace

% Eluent C

% Eluent D

pH (Calculated)

pH (Measured)

10 0 5.60 5.539 1 6.06 6.018 2 6.52 6.497 3 6.98 6.986 4 7.44 7.505 5 7.90 8.054 6 8.36 8.533 7 8.82 9.012 8 9.28 9.441 9 9.74 9.850 10 10.20 10.22

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Salt Gradient Separation at Multiple pH Conditions on Cation Exchange Column

Increasing pH

pH 5.6

pH 7.5 pH 7.0

pH 6.5

pH 6.0

pH 8.0

pH 8.5

ProPac Elite WCX

0 2 4 6 8 10 12 14 16

0

50A

bsor

banc

e (m

AU

)

Retention time (min)

17

pH Gradient Separation at Multiple Salt Concentration on Cation Exchange Columns

0

60

Retention time (min)

0 2 4 6 8 10 12 14 16

10 mM

90 mM60 mM

30 mM

Abs

orba

nce

(mA

U)

Increasing pHIncreasing Salt

ProPac Elite WCX

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Salt Gradient Separation at Multiple pH conditions on Anion Exchange Column

pH 10.2

pH 9.2

pH 6.4

pH 8.3

pH 7.3

pH 5.6

ProPac SAX-10

0 2 4 6 8 10 12 14

0

400A

bsor

banc

e (m

AU

)

Retention time (min)

Increasing pH

19

Salt Gradient Separation at Multiple pH conditions on HIC Column

Retention time (min)

pH 5.6

pH 6.5

pH 9.3

pH 7.4

pH 8.4

pH 10.2

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

0

75

Increasing pH

MAbPac HIC-ButylA

bsor

banc

e (m

AU

)

20

Workflow Automation

Run all programs in previous tablesNumber of peaks

Determine %C and %Dbased on defined criteria

Determine %B range based on main product peak elution

Generate 5 post-script sequence with conditions determined above

Run 5 post-script sequence

Select best condition

Work flow Condition selection criteria

Peak capacity

Peak to valley ratio

𝑛𝑛′ = 1 +𝑡𝑡𝑔𝑔

𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑀𝑀 × 1.7

ℎ𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎ℎ𝑠𝑠𝑠𝑠𝑎𝑎𝑠𝑠𝑠𝑠

ℎ𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎ℎ𝑎𝑎𝑒𝑒𝑒𝑒

Peak to valley (acidic peak) =

Peak to valley (basic peak) =

ℎ𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎ℎ𝑠𝑠𝑠𝑠𝑎𝑎𝑠𝑠𝑠𝑠

×ℎ𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎ℎ𝑎𝑎𝑒𝑒𝑒𝑒