Development of High Capacity Chromatographic Resins for

Biomolecules on Downstream ProcessOscar Yamasaki, Vincent Lim1, Koji Nakamura, Yuji Kubo, Kosuke Araki,

Hiroyuki Moriyama and Shigeru Nakatani, Bioscience Div., TOSOH Corporation, Japan, 1Bioscience Div., TOSOH Asia Pte. Ltd, Singapore

2

Contents

General information• Purification Steps• Impact for cost

Properties on polymer resins• TOYOPEARL® and TSK-GEL® properties• Optimization for high capacity Resin• Ion-exchangers/Mixed-mode chromatography• Hydrophobic Interaction chromatography• Protein A-Affinity chromatography

3

Intermediate step Removal of Major impurities

(including Flow-through mode)

Capture stepLarge columnRough fractionationHigher flow operation

Polishing step

Purified sample (Purity; 99.9 - 99.99%)

Large

(100-300 um)

Small

(20-50 um)

Culture media

Removal of minor impurities,aggregates

Paricle size of resin

Purification Step for Biopharmaceuticals

Intermediate

(50-150 um)

4

What is Ideal Chromatographic Resin Like?

Rigid（high flow rate,

high throughput）(column length)

Long life (durability)

(COGS)

High Capacity（retention）

5

Impact for Cost Reduction on Chromatographic ProcessResin Capacity (Dynamic Binding Capacity)• 100 – 200 g/L DBC on resin• Selectivity including salt tolerant property• Higher flow rate operation

Target molecule concentration in feedstock (up-stream issue)• High DBC for feedstock;up to 10 g/L target

Column size (resin packed volume)• Significant impact up to 200 L column• Single large column or multiple columns system.

Product recovery in total• Multiple steps in process decrease recovery. • Three to four steps should be the maximum. Two step process ideal.

Resin life (number of use cycles)• Over 100-200 cycles column (resin) life should be required.• Disposable resin without cleaning validation but cost issue remains.

Elution volume of target molecule from column (dilution)• The following step takes longer time for operation.

6

Chromatographic Polymethacrylate Resin(Toyopearl® and TSK-GEL®)

Resins for purification of biopharmaceuticals• Protein, peptide, nucleotide, saccharide, API, food additives• IgG, Factor VIII, insulin, oligoDNA, etc.

Rigid, easy to handle and packing• Hydrophilic polymethacrylate polymer resin• Applicable for large column (200 cm I.D.) on TOYOPEARL®

High resolution separation• TOYOPEARL®: particle size from 20 to 200 micron• TSK-GEL®: particle size of 20 and 30 micron

Many separation modes and grades• SEC, IEC, HIC, AFC, (HCIC)

Chemically stable/CIPRegulatory support

• Drug Master File for FDA• Production capacity; ca. 100,000 L/Y

7

Functional group： Simple modification Graft modification Network modification

Binding capacity： Not high high High

Resin shrinkage： insignificant some case ocurred insignificant

Column pressure： Low slightly high Low

High Capacity by Grafting Modification of Functional Groups on Resin

Protein

Functional

group

Simple modification Graft modification Network modification

DEAE-650M GigaCap Q-650, Q-600C AR SuperQ-650M

8

Eluent flow

Porousmaterial

ProteinsIonic groups

+++

++

+ ++ +

++

++

++++

++

+++++

+

+ + +

+

--

-

-

-

Porousmaterial

++

++

++

++

+

++

++

++++

++

+++++

+

+ + +

+

++

+

Ionic groups

-

--

-

-

Proteins

Eluent flow

-

-

--

-

-

--

--

Pore size Small LargeSurface area Large Small“Effective” surface area

for small proteins Large Smallfor large proteins Small Large

High Capacity by Pore Size Technology

9

Pore Size OptimizationPore Size and Adsorption Capacity on Ion-Exchanger

0

20

40

60

80

100

120

3.0 4.0 5.0 6.0

Exclusion limit (Log Mw)*

Bin

ding

cap

acity

for

cyto

chro

m C

(g/L

)

0

10

20

30

40

50

60

Binding capacity for

BS

A (g/L)

Cytochrome C (SP)

BSA (DEAE)

Bovine Serum Albumin (DEAE-type packings, 40-90 mm)Cytochrome C (SP-type packings, 40-90 mm)

Binding capacity was calculated from the absorbance at 280 nm of the eluted protein.*Exclusion limit : determined with PEO

HW-55 HW-65Base resin

10

Pore Size OptimizationPore Size and Adsorption Capacity on HIC Resins

0.0

5.0

10.0

15.0

20.0

25.0

30.0

1.0E+05 1.0E+06 1.0E+07

Exclusion Limit （Log Mw of Pul lulan）

Bin

din

g C

apacit

y f

or γ-

Glo

bulin

s

(mg/m

Ｌ-gel)

HW-55 HW-60 HW-65 HW-75Base resin

11

TOYOPEARL® High-Capacity Resins

(Ion-Exchange and Mixed-Mode Chromatography)Pore Particle sizesize (micron)

TOYOPEARL GigaCap Q-650M AEX medium 75 IgGTOYOPEARL GigaCap DEAE-650M (R&D) AEX medium 75TOYOPEARL SuperQ-650S, M, C AEX medium 30, 60, 100TOYOPEARL Q-600C AR AEX medium 75 Aggregate, Alkaine stableTOYOPEARL Aminated-750F, -650M (R&D) AEX medium 60 Aggregate, salt tolerantTOYOPEARL Q-750M (R&D) AEX large 60 IgMTOYOPEARL DEAE-650S, M, C AEX large 30, 60, 100 Factor VIIITSKgel SuperQ-5PW(20), (30) AEX medium 20, 30 OligoDNATSKgel DEAE-5PW(20), (30) AEX large 20, 30TOYOPEARL GigaCap S-650M CEX medium 75 IgGTOYOPEARL GigaCap CM-650M CEX medium 75 IgG, salt-tolerantTOYOPEARL Sulfate-650M (R&D) CEX medium 60 Aggregate, salt tolerantTOYOPEARL SP-650S, M, C CEX large 30, 60, 100TOYOPEARL CM-650S, M, C CEX large 30, 60, 100 Antibiotics, peptideTOYOPEARL SP-550C CEX small 100 Small protein, peptideTOYOPEARL MegaCap II SP-550EC CEX small 200 Insulin, small proteinTSKgel SP-3PW(30) CEX small 30 Insulin, small proteinTSKgel SP-5PW(20), (30) CEX large 20, 30

TOYOPEARL MX-Trp-650M Mixed medium 60 IgG, salt-tolerant

Ion-exchanger/Mixed-mode Specific applicationType

12

TOYOPEARL® High-Capacity Resins

(HIC and Affinity Chromatography)Pore Particle sizesize (micron)

TOYOPEARL PPG-600M HIC medium 60 IgG, hydrophobic proteinTOYOPEARL Butyl-600M HIC medium 60 IgGTOYOPEARL Phenyl-600M HIC medium 60 IgGTOYOPEARL Phenyl-650S, M, C HIC large 30, 60, 100TOYOPEARL Phenyl-750M (R&D) HIC large IgMTOYOPEARL Butyl-650S, M, C HIC large 30, 60, 100TOYOPEARL SuperButyl-550C HIC small 100 Small proteinTOYOPEARL Ether-650S, M HIC large 30, 60 IgM, hydrophobic proteinTOYOPEARL Hexyl-650C HIC large 100 Hydrophilic protein, plasmidTSKgel Phenyl-5PW(20), (30) HIC large 20, 30TSKgel Ether-5PW(20), (30) HIC large 20, 30 Hydrophobic protein

Hydrophobic interaction chromatography Type Specific application

Pore Particle sizesize (micron)

TOYOPEARL AF-rProtein A-650F AFC large 45 IgG, IgMTOYOPEARL AF-Heparin HC-650M AFC large 60 Coagulation factorTOYOPEARL AF-Blue HC-650M AFC large 60 Nucleotide dependent proteinTOYOPEARL AF-Red-650M AFC large 60 Nucleotide dependent proteinTOYOPEARL AF-Chelate-650M AFC large 60 His-tag proteinTOYOPEARL AF-Epoxy-650M AFC large 60 Activated resinTOYOPEARL AF-Tresyl-650M AFC large 60 Activated resinTOYOPEARL AF-Formyl-650M AFC large 60 Activated resinTOYOPEARL AF-Amino-650M AFC large 60 Activated resinTOYOPEARL AF-Carboxy-650M AFC large 60 Activated resin

TypeAffinity chromatography Typical application

For AFC, high capacity was achieved by optimization of pore size, modification, spacer arms, ligand density and particle size.

13

High Capacity Ion-Exchangers

14

DBC for BSA on Various Anion-Exchangers

Ion-exchanger Particle IEC capacity DBC（BSA）

Recovery

（μm） （eq/L） （g/L-gel） (％)

TOYOPEARL® GigaCap Q-650M 50-100 0.16 175 98

TOYOPEARL® SuperQ-650M 40-90 0.12 149 98

TOYOPEARL® DEAE-650M 40-90 0.11 25 97

TOYOPEARL® Q-600C AR 50-150 0.18 100 98

TOYOPEARL® QAE-550C 50-150 0.33 70 98

Q Sepharose XL 50-150 ＮA 220 96

Capto Q 90 NA 166 98

Fractogel TMAE HiCap 40-90 NA 138 95

Eluent；0.05 mol/L Tris-HCl (pH 8.5) Flow rate； 212 cm/hr, DBC calculated by capacity at 10% breakthrough curve.

15

DBC on Anion-Exchagersfor Proteins with Different Molecular Mass

DBC conditions ; Column; 6 mm I.D. x 4 cmFlow rate; 212 cm/hr, Detection; UV at 280 nmSample; BSA; see Table on page 8 Protein sample; 1 g/L,

human IgG : Loading buffer 0.015 mol/L Tris-HCl buffer (pH8.7) thyroglobulin : Loading buffer 0.015 mol/L Tris-HCl buffer (pH8.7)+ 0.15 mol/L NaCl

TOYOPEARL® GigaCap Q-650M shows the highest capacity for all proteins.TOYOPEARL® Q-600C AR shows the second highest capacity for IgG, while TOYOPEARL® SuperQ-650M shows the second highest capacity for BSA.

BSA human IgG Thyroglobulin66 kDa 160 kDa 660 kDa

TOYOPEARL® Q-600C AR 108 90 26TOYOPEARL® GigaCap Q-650M 173 108 71TOYOPEARL® Super Q-650M 145 13 3TOYOPEARL® QAE-550C 29 32 6TOYOPEARL® DEAE-650M 25 31 3

Binding capacity (g/L-gel)Resin

16

Protein Selectivity on Various Strong Anion-Exchangers

010

203040

5060

7080

0 10 20 30 40 50 60Retention time(min.)

mV

Capto QQ Sepharose XLQAE-550CGigaCap Q-650MQ-600C AR

Conditions;Column size: 7.5 mm I.D. x 7.5 cmElution; Buffer A: 0.05 mol/L Tris-HCl buffer (pH 8.5)

Buffer B: 0.05 mol/L Tris-HCl buffer + 0.5 mol/L NaCl (pH 8.5)Gradient: 60-min linear gradient from buffer A to buffer B

Flow rate:1.0mL/minDetection: UV at 280nmSample: 1. ovalbumin (2.0mg/mL), 2. soybeen trypsin inhibitor (2.0 mg/mL)

Injection volume: 100µL

17

0

20

40

60

80

100

0 5 10 15 20 25 30Retention time (min.)

mV

TOYOPEARL Q-600C AR OriginalTOYOPEARL Q-600C AR 28days

TOYOPEARL Q-600C AR 66daysTOYOPEARL Q-600C AR 106days

1 mol/L NaOH OvalbuminTrypsin inhibitor

Protein Retention after 1 mol/L NaOHStorage on TOYOPEARL Q-600C AR

18

Comparison of IgG Binding Capacityon Various Strong Cation-Exchangers

Ion-ExchangerParticle

sizeIon-exchange

capacity

Dynamic Binding Capacity

(IgG)

Recovery

（μm） （meq/mL） （mg/mL-gel） (％)

TOYOPEARL GigaCap S-650M 50-100 0.16 145 98

TOYOPEARL SP650C 50-150 0.12 12 98

TOYOPEARL SP550C 50-150 0.13 14 98

SP Sepharose XL 50-150 0.17 140 98

Capto S (90) 0.13 138 ND

Fractogel SE HiCap 40-90 0.08 68 97

Flow rate; 212 cm/hr, DBC was calculated by 10 % height of Breakthrough curve.

19

High Flow Rate Capture of Mab Feedstock on TOYOPEARL GigaCap S-650M

Ref.; B. Lain et al., BioProcess International 7(5) (May, 2009), data permitted

Target HCP reduction; < 6.5 ug/mg of Mab

No significant difference in HCP reduction at sample loading conc. at 7.7 g/L, while DBC decreased to 79 g/L.

20

DBC for PEGylated Lysozymeon Strong Cation-Exchangers

Lysozyme mono-PEG5Lysozyme

mono-PEG30Lysozyme

TSKgel SP-5PW (20) 20 33 28 15TOYOPEARL SP-650M 65 28 23 10TOYOPEARL SP-550C 100 69 53 N/ATOYOPEARL MegaCap II SP-550EC 200 51 28 N/ATOYOPEARL GigaCap S-650M 75 140 95 5

Conditions Column size; 1 mL Buffer; 25 mM sodium phosphate buffer (pH 6.0) PEG; PEG5 (MW; 5 KDa), PEG30 (MW; 30KDa)

Particlesize (um)

Dynamic Binding Capacity (DBC; g/L)Resin

Ref.; A. Moosman et al., J. Chromatogr.A 1217 (2010) 209-215

Lysozyme

5KD 30KDPEG PEG

Lysozyme; MW14KDa

21

Peak Width for Elution of Protein (Mass Transfer) by GigaCap S-650M

-200

0

200

400

600

800

1000

1200

75 125 175 225

Time（min.）

mV

GigaCap S-650M（ｈIgG）

Capto S（ｈIgG）

22

Comparison of DBC for Human IgGon Weak Cation-Exchangers

Conditions;Dynamic binding capacities were determined at 10% breakthroughColumn size: 6 mm I.D. x 4 cmSample: polyclonal human IgG (1 g/L)Loading buffer 0.05 mol/L sodium acetate buffer (pH 4.7)Elution buffer 0.05 mol/L sodium acetate buffer (pH 4.7) + 0.5 mol/L NaClLinear velocity: 212 cm/hr, Detection: UV @ 280 nm

TOYOPEARL® GigaCap CM-650M has the highest DBC for IgG.

(μm) (eq/L) (g/L-gel) (%)

TOYOPEARL® GigaCap CM-650M 50-100 0.24 100 98

CM Sepharose Fast Flow 45-165 0.09-0.13 22 93

Fractogel EMD COO-　(M) 40-90 - 37 97

RecoveryIon Exchanger Particle sizeIon

exchangecapacity

Dynamicbindingcapacity

23

Comparison of Mixed-Mode Resins(Salt-tolerant Separation)

DBC(IgG)

Recovery DBC(IgG)

Recovery

TOYOPEARL MX-Trp-650M 50 - 100 0.12 95 g/L 97% 48 g/L 96%Capto MMC mean75 0.07 14 g/L 86% 11 g/L 85%Binding Buffer; 0.05 mol/L acetate buffer (pH 4.7) + 0.1 mol/L (conductivity; 12 uS/mL) 0.05 mol/L acetate buffer (pH 4.7) + 0.15 mol/L (conductivity; 17 uS/mL) Flow rate; 212 cm/hrSample; polyclonal IgGDBC; 10 % peak height on breakthrough curve for IgG

Particlesize(um)

Ion-exchangecapacity

(meq/mL)

Conductivity in Binding Buffer12 mS/cm 17 mS/cm

Mixed-mode resin requires optimization of binding, washing and elution conditionsin terms of combination of salt concentration and pH.

High Capacity HIC and Affinity Resins

25

Diversity of Hydrophobicity on Monoclonal Antibody

(Retention on HP-HIC on TSKgel Phenyl-5PW)

HPLC ConditionColumn : TSKgel Phenyl-5PW (7.5 mm I.D. x 7.5 cm)Eluent : (A) 0.1 mol/L phosphate buffer containing 1.8 mol/L ammonium sulfate (pH 7.0)

(B) 0.1 mol/L phosphate buffer (pH 7.0)Gradient : (B)0%(0min)--0%(5min)--100%(65min) linear Flow Rate : 1 mL/minSamples : 62 mouse monoclonal antibodies; IgG1, IgG2a, IgG2b, IgM

0

10

20

30

40

50

60

70

80

90

No.

1

No.

3

No.

5

No.

7

No.

9

No.

11

No.

13

No.

15

No.

17

No.

19

No.

21

No.

23

No.

25

No.

27

No.

29

No.

31

No.

33

No.

35

No.

37

No.

39

No.

41

No.

43

No.

45

No.

47

No.

49

No.

51

Antibody

Elu

tion

time

of e

ach

antib

ody

on

TSK

gel p

heny

l-5P

W(m

in)

IgG2b

IgM

IgG2aIgMIgM

IgG2a

IgG2b

IgG2b

IgG2b

◆ IgG1

26

TOYOPEARL HIC Ligand Spectra

Phenyl

O

Ether

(OCH2CH2)nOH

Butyl

OOH

n

PPG

IncreasingHydrophobicity Hexyl

O(CH2)5CH3

O(CH2)3CH3

Pore size grade600, 650, 750(R&D)

550, 600, 650, 800 (R&D)

Ether; Oligoethylene glycol

PPG;Polypropyrene glycol

27

Dynamic Binding Capacity on HIC Resins

Gamma-Globulin (IgG) Lysozyme

DBC(g/L)

Recovery(%)

DBC(g/L)

TOYOPEARL Phenyl-600M 35 93 58

Phenyl-Sepharose High Sub 32 87

87

95

ND

52

TOYOPEARL Butyl-600M 29 ND

TOYOPEARL Phenyl-650M 16 27

TOYOPEARL PPG-600M 3 ND

Conditions;IgG; 0.8 mol/L ammonium sulfate in 0.1 mol/L sodium phosphate buffer (pH 6.8)Lysozyme; 1.8 mol/L ammonium sulfate in 0.1 mol/L sodium phosphate buffer (pH 6.8)

Phenyl-600M showed high capacity and recovery.

28

Efficiency of Purification Cycle by HIC for Humanized Monoclonal Antibody(Model Case with Purified Sample)

Recovered Recoveredcapacity*1/cycle Sample Desorption*3 Re- capacity/time

(g/L/cycle) (%) charge (recovery) equilibration (g/L/hour)Phenyl-650M 34.5 98 141 16 20 177 11.7Butyl-650M 36.6 87 168 49 20 237 9.3PPG-600M 46.1 103 179 8 20 207 13.4Butyl-600M 44.6 92 194 44 20 258 10.4

*1; Antibody eluted was fractionated and determined with UV detector*2; Recovery means Recovered capacity/Dynamic binding capacity (10% leakage)*3; Until reaching 10 % of feed concentrationPurification cycle is from equilibration, feed to recovery.Column size; 7.8 mm I.D. x 20 cm Flow rate; 300 cm/hr Sample; humanized IgG (1 g/L)Buffer; 31 mmol/L citrate, 69 mmol/L phosphate buffer (pH 6.6) containing 1.0 mol/L ammonium sulfateEquilibration is proceeded with 5 CV

TOYOPEARLCycle time (min)

TotalRecovery*2

29

Selectivity of Protein Separation on HIC Resins

0 10 20 30 40 50 60 70

Elution volume [mL]

UV

at 2

80 n

m

1. Ribonuclease A2. Lysozyme3. α-Chymotripsinogen A

１

2

3

Phenyl-600M

Phenyl-Seph FF(High Sub)

30

TOYOPEARL AF-rProtein A-650F

Alkaline durable resin•0.1 mol/L NaOH, 120 CIP, no DBC change•0.5 mol/L NaOH, 50 CIP, 10 % DBC decrease

High capacity at higher flow rateRigid resin applicable to large column

Support matrix TOYOPEARL® HW-65Particle size (micron) 50 - 100Pore size (nm) 1000Immobilized ligand recombinant protein A (E. Coli)Ligand coupling method multi-point attachmentStatic binding capacity (g/L) 52Dynamic binding capacity* (g/L) > 30* Residence time of 2 min

30-50100

31

DBC Comparison at Various Residence Time

15

20

25

30

35

40

45

0 0.5 1 1.5 2 2.5 3 3.5

Residence Time(min)

IgG

DB

C(m

g/m

L)

改良65F

65M

MabSel SuRe

New type

TOYOPEARL AF-rProtein A-650F showed 20-50 % higher DBC at residence time less than 2 min compared to MabSelect® SuRe

32

Column Length and Flow rate on Productivity

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0 200 400 600 800 1000

Sample loading linear velocity (cm/h)

Pro

ductivi

ty (

g/m

in)

10cm

20cm

30cm

Column size; TOYOPEARL AF-rProtein A-650F 10 mm I.D. x 10 cmPre-equilibration; 3 CV with 0.02 mol/L sodium phosphate buffer (pH 7.4) containing 0.15 mol/L NaClSample loading; IgG, 1 g/LFlow rate; various flow rateElution; 3 CVCleaning; 1 CV with acidic buffer, 16 min with 0.1 mol/L NaOH, 3 CV with water

30 cm column length showed the highest productivity for IgGin sample loading flow rate over 300 cm/hr.

33

Conclusion•High binding capacity for proteins was obtained by modification of hydrophilic polymethacrylate resin for IEC, mixed-mode and HIC resins.• High capacity of ion-exchangers and mixed-mode resin was mainly achieved by grafting modification of functional groups. Those ion-exchanges showed high binding capacity for various proteins even with different molecular mass. •High capacity of HIC resins was obtained mainly by optimization of pore size of resin for target protein with different molecular mass.•Larger pore and rigid resins could perform higher production efficiency at higher flow rate or lower residential time.

34

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