chromatographic media for antibody / protein purification · chromatographic media for antibody /...

2011 Mitsubishi Chemical Corporation All Rights Reserved.

Chromatographic Media for Antibody / Protein Purification

Separation Materials Department

Mitsubishi Chemical Corporation (MCC)


Technology to control physical properties, such as pore radius, distribution, functionality and surface area

Variety of Installation Records Installed volume >2,200m3 (export 1,400m3, domestic 800m3, in past 5 yrs) Customers: Major pharmaceutical companies (in JPN, US, India, UK, Germany, FR, Switzerland, Ireland, etc)

Major applications:

Human Insulin Lisinopril Cephalosporin C Vancomycin Daptomycin Cefotaxime Imipenem

New: Antibody/ Protein

P.2

-

100,000

200,000

300,000

400,000

500,000

2007 2008 2009 2010 2011 2012

Export Domestic Total

Vo

lum

e (L)

year

Background: MCC Bioseparation Media


P.3

Peptides, Proteins MW 1000-100,000 Macrolides

MW 50- 1500

10 100 1000Pore size ()

SP825L HP21

SP850

SP70 SP700

HP20

SP207

-Lactums MW ~600

HP2MGL

600-800

1,000-2,000

4,000-5,000

(Reference) Ability to control media properties

Example) Synthetic Adsorbent SEPABEADSTM


P.4

Protein A affinity chromatography

MabSpeedTM rP series

Ion-exchange chromatography

ChromSpeedTM series

type S strong cation exchanger

type Q strong anion exchanger

type CM weak cation exchanger

type DA weak anion exchanger

--- New Media for Biopharmaceutical industry --- Highly productive & efficient purification via high throughput!

NEW ADDITION to the family!!!


P.5

--- New Production Facility for Bio-separation Media ---

Built in February 2013

Prepared in clean dedicated facility

Hygiene management by ISO9001


P.6

--- Product Lineup for MCC Bio-separation Media---

Affinity Ion Exchange

Product MabSpeedTM ChromSpeedTM

rP S Q CM DA

Matrix Crosslinked Polymethacrylate

Ligand / Functionality

rProtein-A -SO3- -N+(CH3)3 -COO

- -N+H(CH3)2

Particle Size (m)

35, 45 30, 60


P.7

Spherical and mono-disperse particles

Easy and reproducible packing

Low pressure drop

Non-compressive bed

Rigid and durable matrix

No volume change

Longer life

Wide pH & solvent compatibility

MCCs New Bio-separation Media---

High performance for achieve high throughput purification


Production/Sales

Mitsubishi Chemical Co. (Japan)

Tai Young Chemical Co., Ltd.

Resindion (Italy)

Sales Mitsubishi Chemical China Commerce Ltd. Mitsubishi Chemical India Pvt. Ltd. ITOCHU Chemicals America Inc.

Resindion

TYC

MCC ICAI MCN

MCI

Sales/Production network

SYFT


Affinity chromatography MabSpeedTM

With wild type ligand: MabSpeedTM P102 (45 m) & rP111 (35 m) With engineered type ligand: MabSpeedTM P202 (45 m)


P.10

--- MCC Protein-A affinity media line-up--

Mode Protein-A Affinity

Product MabSpeedTM

rP102 rP111 rP202*

Matrix Crosslinked Polymethacrylate

Ligand / Functionality

rProtein-A

(wild type)

rProtein-A

(engineered type)

Particle Size (m) 45 35 45

DBC (g/L-resin)

10% breakthrough,

-globulin, r.t. 3min)

24 33 >50

*Matrix of MabSpeedTM rP202 has been modified to have even higher DBC with the original ligand. *MabSpeedTM rP202 media/columns are currently avilable in Asia and will be available in other regions in the end of 2016. For details, please inquire us.


P.11

:*Pressure drop data. Data was taken with a condition of a column 20 ID x 200 mm with liquid water at temperature 25 C.

--- Hydraulic data represents low pressure drop & easy packing ---


P.12

MabSpeedTM 0 G MabSpeedTM 5,000 G

Polysaccharide 0 G Polysaccharide 5,000 G

reversible

irreversible

Pressure durability


600

GE MabSelect SuRe

200

400

200 400

200

MabSpeedTM rP102

MabSpeedTM rP111

DBC (mg-IgG/mL-resin)

A2

80

(C/C

0, %

) Breakthrough profiles at various flow rate

(cm/h) 200 400 600 MabSelect SuRe

42.3 32.7 -

MabSpeedTM

rP111 35.6 32.5 30.4

MabSpeedTM

rP102 26.1 24.0 16.2

Dynamic binding capacity

Column 5mm ID x 200 mm Sample: 1.0 mg/mL human -globulin Buffer: PBS pH 7.4, Temp: 20 deg C The DBC was determined at 10% breakthrough. Flow: 200, 400, 600 cm/h

(10% BTC)

Fast kinetics, both fast adsorption and desorption (later discussed) leads to shorter operation time; high IgG production!


Wild type ligand: MabSpeedTM P102 (45 m)

& MabSpeedTM rP111 (35 m)


mAb purification from cell culture

Conditions: Column: MabSpeed rP111 5mm I.D. x 20cm. (BV:4.0mL ) Binding buffer PBS Wash buffer PBS Elution buffer 0.1M Citrate pH3.0 Flow rate 400cm/h System AKTA avant Sample CHO cell culture containing 0.1mg/mL mAb, 100mL

mL

A2

80

>HCP contamination Start material: 116,160 ppm(ng-HCP/mg-IgG) (HCP: 11,616 ng/mL IgG: 0.1mg/mL) IgG fraction - MabSpeedTM rP111: 9.2 ppm(ng-HCP/mg-IgG) - MabSelect SuRe 63.7 ppm >PrA ligand leakage - MabSpeedTM rP111: 3.4 ppm (ng-PrA/mg-IgG) >Recovery - >98% (UV280nm)

1 Molecular weight marker 2 start material 3 path through 4 mAb fraction

1 2 3 4


P.16

CIP durability

DB

C(%

) P

rA leak (p

pm

)

5.62

0.25

cycles Column: 5mm ID x 5cm Binding: Phsphate bufferd saline(pH7.4) Elution: 0.1M Sodium Citrate pH3.0 CIP: 0.1M NaOH Contact: 15min


P.17

@ pH 3.0

@ pH3.5

MabSpeedTM

(rProtein A) Other polymer media

rP102 data

Spin column 0.5mL resin single cycle (1-6)

1. equilibration buffer A, 6 BV

2. sample load 1mg/mL g-globulin, 2 BV

3. washout buffer A, 6 BV

4. elution buffer B, 4 BV

5. regeneration buffer C, 4 BV

Each Step 500 g x 1 min

buffer A: PBS pH7.4

buffer B: 0.1M sodium citrate pH3.0, 3.5

buffer C: 1M Tris-HCl pH9.0

Agarose (alkali resistant)

Effect of elution pH

- Sharp elution for MabSpeed at pH 3.5 - Elution conditions for wild type of rProtein-A ligand can be applied for MabSpeed.


P.18

Faster equilibrium of MabSpeed contributes buffer and time savings about 30%

Buffer exchanging profiles

MabSpeedTM rP111 MabSelect SuRe

200 400

100 200 400

100 Column : 10 ID x 200 mm Base : 0.8M NaCl Load : 0.05M NaCl, Temp : 20 deg-C Flow : 100, 200, 400 cm/h

(cm/h) 100 200 400

MabSpeedTM rP111 10.7 5.4 2.8

MabSelect SuRe 14.0 7.2 3.8

Time to equilibration (min, ~6.0>mS/cm)

Flow time (min)

Co

nd

uct

ivit

y(m

S/cm

)


MabSelect SuRe

400cm/h

min/cycle

DBC (1% BTC,g/L-resin)

cycle/day

(adsorption) (misc)

IgG Production (g/day)

62 60

11.8

20.6

382

Colume volume (L) 1.57

flow rate (cm/h)

Column height (cm)

Productivity Simulation

MabSpeed rP111

400cm/h 600cm/h

89 48

10.5

29.8

56 37

15.4

28.2

489 681

1.57 1.57

200cm/h

200 105

4.7

33.3

247

1.57

200cm/h

206 81

5.0

34.4

270

1.57

MabSpeedTM rP111 results in higher productivity than MabSelect SuRe


P.20

Mab purification Chromatography with MCC resins

Protein A affinity : MabSpeedTM rP111

Cation exchange : ChromSpeedTM S101

Anion exchange : ChromSpeedTM Q101

Column MabSpeedTM rP111 10x150mmH(BV: 12mL) Sample Clarified CHO cell culture, 0.5mg/mL mab (20.8mg/mL-resin) Equiliburation buffer 20mM Sodium Phosphate, 150mM NaCl, pH7.4 Elution buffer 20mM Sodium Citrate, pH3.4 Flow 300 cm/h, R.T. 3min Temp. 20 deg-C

Column ChromSpeedTM S101 5x200mmH(BV: 4mL) Sample mab (51.3 mg/mL-resin) after purification on MabSpeedTM rP111 Start buffer 20mM Sodium acetate, pH5.5 Elution buffer 20mM Sodium acetate, 1M NaCl, pH5.5 Flow 300 cm/h, R.T. 4min Gradient 5% (10CV), 15% (20CV), 100% (10CV) Temp. 20 deg-C

Column ChromSpeedTM Q101 5x50mmH(BV: 1mL) Sample mab (192 mg/mL-resin), eluate after ChromSpeedTM S101, pH adjusted to 6.0 Start buffer 20mM Sodium acetate, pH6.0 Flow 300 cm/h, R.T. 1min Temp. 20 deg-C

capture, removal of aggregate, HCP,

removal of agg, HCP, PrA

removal of DNA, endotoxin,

Cell culture

Three step processAccumulated

yield (%)

Dimers and

aggregate (%)

Protein A

(ppm)

HCP

(ppm)

Start material 100 - - 35717

MabSpeedTM rP111 98


Engineered type ligand: MabSpeedTM P202 (45 m)


High Dynamic Binding Capacity (DBC):

P.22

DBC of MabSpeed rP202, well exceeding those agarose and/ or polymeric media in standard flow rate in 100 cm/hr (r.t of

12 min) to 400 cm/hr.

DBC of MabSpeed rP202: keeps the high DBC even at high folwrate in the range of 600 cm/hr (r.t. of 2 min) to 1,000 cm/hr (r.t. of 1.2

min)

Drawback of agarose media: high pressure drop

(or back pressure) prevents to utilize high flow rates, sometimes resulting very low DBC.

R.T.(min) 1.5 2.0 3.0 6.0

1 g/L IgG 42 44 51 62

5 g/L IgG 38 44 50 59

MabSpeed rP202 DBC

*Data was taken with a column 5 x 200mmH with a sample of 1 mg/mL human IgG. The straight red arrow shows high DBC at high flow rate while the blank green arrow of agarose media shows significant decrease of DBC.


CIP Durability

P.23

Cycles

DB

C (

%)

Lig

an

d le

ak

(pp

m)

*Experiments were performed with a column of 5mm ID x 5cm, by binding with phosphate buffered saline(pH7.4) and eluting with 0.1M sodium citrate at pH3.0. The contact time was 15min.

High DBC and Protein-A ligand leakage (


MabSpeed rP202 new engineered ligand enables

elution with weakly acidic condition of pH 4.0:

P.24

3.9 3.4 3.9

3.9

Numbers in the profiles are pH of eluate pool. Data are taken with a condition of a column 5 x 50 mmH (1mL), flow rate 1.0 mL/min, eluent A 0.1 M, citrate pH6.5, eluent B 0.1 M citrate pH2.5, pH gradient 0->100 %B over 20 CV (20 mL), and with a sample of 20 g/mL mab in TST, 10 mL (0.2 mg):

MabSpeedTM rP111 (wild type ligand)

MabSpeedTM rP202 (engineered type ligand)


CHO cell culture purification

P.25

Low HCP contamination and Protein-A ligand leakage with high recovery

A2

80

mL

>HCP contamination Start material: 116,000 ppm(ng-HCP/mg-IgG)

IgG fraction : 19 ppm(ng-HCP/mg-IgG) (3.8 log down) >Protein A ligand leakage IgG fraction : 6.3 ppm (ng/mg-IgG)

Conditions: Column MabSpeed rP202 26 x 7mm I.D. (BV:1.0mL ) Binding buffer PBS Wash buffer PBS Elution buffer 0.1M Citrate pH3.0 Flow rate 1.0 mL/min System AKTA avant Sample CHO cell culture containing 0.99 mg/mL mAb, 30mL


Buffer Exchange

P.26

Flow time (min)

Co

nd

uct

ivit

y(m

S/cm

)

200 cm/hr

400 cm/hr

100 cm/hr

(cm/h) 100 200 400 600

MabSpeedTM rP202

17.1 8.6 4.4 3.0

MabSelect SuRe

18.8 9.8 5.0 X

Time to equilibration (min, ~6.0>mS/cm)

Column : 10 ID x 200 mm Base : 0.8M NaCl Load : 0.05M NaCl, Temp : 20 deg-C Flow : 100, 200, 400 600cm/h

~ 10% saving of buffers due to fast desorption with MabSpeedTM rP202!


MabSelect SuRe

400cm/h

min/cycle


cycle/day

(adsorption) (misc.)

IgG production (g/day)

98 60

9.1

33

468

Column volume (L) 1.57

flow rate (cm/h)

Column height 20cm

Productivity simulation 1

MabSpeed rP202

400cm/h 600cm/h

153 54

6.9

51

88 42

11.1

44

555 769

1.57 1.57

200cm/h

254 105

4.0

42

267

1.57

200cm/h

372 93

3.1

62

301

1.57

IgG production: MabSpeedTM rP202 well exceeds MabSelect SuRe


MabSelect SuRe LX

400cm/h

121 60

7.9

40

504

1.57

MabSpeed rP202

400cm/h 600cm/h

153 54

6.9

51

88 42

11.1

44

555 769

1.57 1.57

200cm/h

326 105

3.3

54

285

1.57

200cm/h

372 93

3.1

62

301

1.57

Productivity simulation 2

min/cycle


cycle/day

IgG production (g/day)

Column volume (L)

flow rate (cm/h)

Column height 20cm

IgG production: MabSpeedTM rP202 well exceeds MabSelect SuRe LX


Ion exchange chromatography ChromSpeedTM

ChromSpeedTM S103, Q103, CM103, and DA103 (60 m) ChromSpeedTM S101, Q101, CM101, and DA101 (30 m)


P.30

Batch adsorption capacity

Ion exchange capacity

(eq/L-resin)

Batch adsorption capacity

(g/L-resin)

Human -globulin

0.09 125

Functional group

60

Particle diameter

(m)

124 0.08 60

SBC condition: S- and CM- type: Human -globulin 2.5 g/L, 20 mM citrate (pH 5.2), 25 Q- and DA- type Human -globulin 2.5 g/L, 20 mM Tris-HCl (pH 9.0), 25

-N+H(CH3)2

-COO 114 0.11 60

81 0.13 60

0.08 140 30

130 0.08

-N (CH3)3

30

98 0.11 30

120 0.10 30

-SO3

ChromSpeed

S103

S101

Q103

Q101

CM103

CM101

DA103

DA101


P.31

Pressure drop comparison (Water, 25C)

0.00

0.40

0.80

1.20

1.60

2.00

0 500 1000 1500 2000

Linear velocity (cm/h)

Pre

ssu

re d

rop

(M

Pa

/m)

ChromSpeed Q103, 60m

Agarose-based resin 1, 90m

Agarose-based resin 2, 90m

Excellent Hydraulic Property --- No bed compression at 1,800cm/hr linear velocity ---

Hydraulic properties of ChromSpeedTM


1-1) S103 (60m) vs competitors

ChromSpeedTM S103 (Mitsubishi)

UV

28

0n

m

Capto S (GE)

(a) (b) (a) (b)

Conditions: Column 100 x 5mm I.D. (BV:2.0mL ) Eluent A 20mM Sodium Phosphate (pH6.5) Eluent B A + 1.0M NaCl Flow rate 1.0mL/min (R.T. 2min) Gradient 0-100% B over 60min Sample (a) Cytochrome C + (b) lysozyme = 125 / 125ug / 50uL (pI) (9.3) (11.0)

Gigacap S 650M (TOSOH)

(a) (b)

60m 90m 50-100m


Effect of IgG Concentration

P.33

Column 5 x 100 mmH (BV:2.0mL) Buffer 20mM Sodium Acetate (pH5.5) Flow rate 200, 400, 600, 800 cm/h (R.T. 3.0, 1.5, 1.0, 0.75 min) Sample a) 1.0, b) 2.5, c) 10 mg/mL gamma-globulin (IgG)

a) 1.0 mg/mL b) 2.5 mg/mL

10

% D

BC

(m

g-Ig

G/m

L-re

sin

)

c) 10 mg/mL

The highest dynamic binding capacity was observed for ChromSpeedTM S103. Moreover, DBC showed less dependence on the IgG concentration for ChromSpeedTM S103.


Durability of ChromSpeedTM S103

SBC *1 >120 mg-IgG/mL-resin Recovery*2 >99% (IgG) Pressure Drop*3 < 1.5MPa/m at 1,000 cm/h Alkaline tolerance*4 100cycles of 0.5M NaOH exposure O.K.

*1 2.5 g/L, 20 mM Sodium acetate (pH 5.5), 20 deg-C *2 1.0 g/L, 20 mM Sodium acetate (pH 5.5), 20 deg-C *3 10x200mmH, 150mM NaCl, room temperature *4 0.5M NaOH, 15min/cycle, 20deg-C, DBC: IgG at 10 % breakthrough

cycles

DB

C(%

)



UV

28

0n

m

(a) (b)



(a) (b)

1-2) S103 (60m) vs S101 (30m)

60m 30m




Capto SP ImpRes (GE)

UV

28

0n

m

(a) (b)

(a) (b)

1-3) S101 (30m) vs competitors

36-44m 30m


ChromSpeedTM Q103 (Mitsubishi)

UV

28

0n

m

Gigacap Q (TOSOH)

(a)

(b)

(min) (min)

(a)

(b)

Conditions: Column 100 x 5mm I.D. (BV 2mL) Eluent A 50mM Tris-HCl (pH8.5) Eluent B A + 1.0M NaCl Flow rate 1.0mL/min (R.T. 2min) Gradient 0-100% B over 60min Sample (a) Myoglobin + (b) Trypsin inhibitor = 250/50ug / 25uL (pI) (7.5) (4.5)

2-1) Q103 (60m) vs competitors

60m 50-100m



UV

28

0n

m

(a)

(b)


(min) (min)

(a)

(b)


2-2) Q103 (60m) vs Q101 (30m)

60m 30m


Capto Q ImpRes (GE)

UV

28

0n

m


(min) (min)

(a)

(b)

2-3) Q101 (30m) vs competitors

(min)

(a)

(b)


30m 36-44m


UV

28

0n

m


GigaCap CM 650M (TOSOH)

(a) (b)

ChromSpeedTM CM103 (Mitsubishi)

(a) (b)

3-1) CM103 (60m) vs competitors

Comparable product unavailable

Capto CM (GE)

UV

28

0n

m

60m 60m


UV

28

0n

m

(a) (b)




(a) (b)

3-2) CM103 (60m) vs CM101 (30m)

UV

28

0n

m

60m 30m


P.42

4-1) DA103 (60m) vs competitors

ChromSpeed-DA103 Agarose-based DEAE medium

Conditions: Column, 100 x 9mmI.D. (6.4ml); Conditions: Column, 5ml;

Black Eluent A, 20mM sodium phosphate (pH7.0); Black Eluent A, 20mM sodium phosphate (pH7.0);

Eluent B, A + 1.0M NaCl; Eluent B, A + 1.0M NaCl;

Blue Eluent A, 20mM Glycine-NaOH (pH10.0); Blue Eluent A, 20mM Glycine-NaOH (pH10.0);

Eluent B, A + 1.0M NaCl; Eluent B, A + 1.0M NaCl;

Flow rate, 1.0ml/min (94cm/h); Gradient, 0-50% B over 30min. Flow rate, 0.786ml/min; Gradient, 0-50% B over 30min.

Samples: a-Lactalbumin, 160mg / 160ml Samples: a-Lactalbumin, 125mg / 125ml

-9.00E+03

0.00E+00

9.00E+03

1.80E+04

2.70E+04

3.60E+04

4.50E+04

5.40E+04

6.30E+04

7.20E+04

8.10E+04

0 5 10 15 20 25 30 35 40

time (min)

UV

28

0n

m (m

V)

-9.00E+03

0.00E+00

9.00E+03

1.80E+04

2.70E+04

3.60E+04

4.50E+04

5.40E+04

6.30E+04

7.20E+04

8.10E+04

0 5 10 15 20 25 30 35 40

time (min)

UV

28

0n

m (m

V)

pH7pH10

pH10pH7

Capto DEAE (GE)

ChromSpeed DA103 (Mitsubishi)

60m 90m


UV

28

0n

m

ChromSpeedTM DA101 (Mitsubishi)

ChromSpeedTM DA103 (Mitsubishi)

4-2) DA103 (60m) vs DA101 (30m)

Conditions: Column, 50 x 5mmI.D. (60mm); Eluent A, 20mM sodium phosphate (pH7.0); Eluent B, A + 1.0M NaCl; Flow rate, 0.5ml/min (150cm/h); Gradient, 0-50% B over 30min. Samples: a-Lactalbumin / Trypsin inhibitor = 25mg / 50mg / 25ml

60m 30m


Product Details

Standard package size: 25 / 100 / 1000 mL Slurry in 20%-Ethanol

Documentations

COA MSDS RSF (NDA required)

Screening columns available for ChromSpeedTM 103 Series 1 mL columns 5 mL columns


Thank you


Reference


ChromSpeedTM S103 (60um, Mitsubishi)

ChromSpeedTM S101 (30um, Mitsubishi)

Capto S (90um, GE healthcare)

Capto SP ImpRes (40um, GE healthcare)

GigaCap S650M (50-100um, TOSOH)

CellufineMax S (40-130 um, JNC)

Nuvia S (85um, Bio-Rad)

UV

28

0

time(min)


GigaCap S650S (20-50um, TOSOH)

SP Sepharose FF (90um, GE healthcare)

BioPro S75 (75um, YMC)

Eshmuno S (75-95um, Merck Millipore)

-50000

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

0 10 20 30 40

Selectivity of Strong Cation Exchangers


-10000

0

10000

20000

30000

40000

50000

60000

70000

0 5 10 15 20 25 30 35 40ChromSpeed Q103 (60um, Mitsubishi)

ChromSpeed Q101 (30um, Mitsubishi)

Gigacap Q650M (50-100um, TOSOH)

Gigacap Q650S (20-50um, TOSOH)

Q Sepharose FF (90um, GE healthcare)

Capto Q (90um, GE healthcare)

CaptoQ ImpRes (40um, GE healthcare)

time(min)

Conditions: Column 100 x 5mm I.D. (BV:2.0mL ) Eluent A 50mM Tris-HCl (pH8.5) Eluent B A + 1.0M NaCl Flow rate 1.0mL/min (R.T. 2min) Gradient 0-100% B over 60min Sample alpha lactalbumin + Trypsin inhibitor = 25 / 125ug / 25uL (pI) (4.4) (4.5)

UV

28

0

Selectivity of Strong Anion Exchangers

chromatographic media for antibody / protein purification · chromatographic media for antibody /...

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