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Making the Impossible Possible –

Chromatographic Solutions for Demanding

Separations in Downstream Processing

BioInnovation Leader Summit 2015 Judith Vajda, Regina Römling, Egbert Müller

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•  Many mAb purification processes are based on 3 chromatographic platforms

•  Is it possible to set up a mAb purification process based on 2 chromatographic platforms?

impossible

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Outline

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•  mAb platform approach

•  Requirements and solutions for mAb capturing in a 2-step scenario

•  mAb Polishing within one chromatographic step

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Common purification processes consist of 3

platforms

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Primary Recovery

Protein A Capturing CEX AEX Final

Filtration

Primary Recovery

Protein A Capturing HIC IEX Final

Filtration

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Purification requirements

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•  HCP reduction to ppm level

•  Low Protein A leaching

•  Aggregate removal

•  cellular DNA <100 pg per dose •  Viral clearance should be considerably higher than the

potential content in the source material

Source: Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/OtherRecommendationsforManufacturers/UCM153182.pdf Birch, John R.; Racher, Andrew J.; Antibody production. Advanced Drug Delivery Reviews 58 (2006) 671-685.

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Capturing - Protein A chromatography

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•  High capacity allows product concentration

•  Extensive HCP removal

•  Low protein A leakage

Capturing with HC Protein A

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High capacity Protein A dynamic binding capacity

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TOYOPEARL AF-rProtein A HC-650F shows high capacity for mAbs. Efficient adsorption of feed concentrations as high as 15 g/L

IgG A mAb B

0.8 min res. time

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HCP log reduction value

HCP log reduction values greater than 3 are possible!

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

LOG

uni

ts o

f CH

OP

redu

ctio

n

no. of experiment

TP AF rProteinA HC 650F (Citrate) TP AF rProteinA HC 650F (Acetate)

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Titer dependency – CHOP content

High titer feedstreams improve CHOP clearance of protein A chromatography!

% Spiking (v/v) with 10 x concentrated CCF

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Protein A leakage

Protein A leakage of TOYOPEARL AF-rProtein A-HC 650F is smaller than 10 ng/ml.

Design-Expert® SoftwareProteinA leaching

10

0

X1 = A: pHX2 = B: load

Actual FactorsC: titer = 4.75D: spiking = 15.00

2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25

20.00

25.00

30.00

35.00

40.00ProteinA leaching

elution pH

colu

mn

load

[mg/

mL]

2

2

468

40.0

35.0

30.0

25.0

20.02.252.502.753.003.253.503.754.004.25

68 24

Design-Expert® SoftwareProteinA leaching

10

0

X1 = A: pHX2 = B: load

Actual FactorsC: titer = 4.75D: spiking = 15.00

2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25

20.00

25.00

30.00

35.00

40.00ProteinA leaching

elution pH

colu

mn

load

[mg/

mL]

246

8

2

Design-Expert® SoftwareProteinA leaching

10

0

X1 = A: pHX2 = B: load

Actual FactorsC: titer = 4.75D: spiking = 15.00

2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25

20.00

25.00

30.00

35.00

40.00ProteinA leaching

elution pH

colu

mn

load

[mg/

mL]

246

8

40.0

35.0

30.0

25.0

20.02.252.502.753.003.253.503.754.004.25

6

8

24

Design-Expert® SoftwareProteinA leaching

10

0

X1 = A: pHX2 = B: load

Actual FactorsC: titer = 4.75D: spiking = 15.00

2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25

20.00

25.00

30.00

35.00

40.00ProteinA leaching

elution pH

colu

mn

load

[mg/

mL]

246

8

Citrate buffer Acetate buffer

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How can we eliminate one chromatographic step?

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Primary Recovery

Protein A Capturing CEX AEX Final

Filtration

Primary Recovery

Protein A Capturing HIC IEX Final

Filtration

Primary Recovery

Protein A Capturing AEX Final

Filtration

Aggregates?

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Retention of BSA on Various AEX Resins

• Column: TOYOPEARL NH2-750F, 5 mm ID×5 cm (1 mL), • Mobile Phase A: 20 mmol/L Tris-HCl (pH 8.0), • Mobile Phase B: Buffer A + 2.0 mol/L NaCl (pH 8.0) • Gradient: 0-100% B, 120 min linear gradient • Flow-rate: 1.0 mL/min, Detection: UV @ 280 nm • Sample : BSA 1 mg

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NH2-750F shows higher retention than other AEX media

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pH Dependence of Elution of BSA

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Column: TOYOPEARL NH2-750F, 5 mm ID×5 cm (1 mL), Mobile Phase A: pH 4.5 & pH5: 30 mmol/L N-methyl piperazine

pH 6.0: 20 mmol/L Bis-Tris pH 7.5: 20 mmol/L Tris-HCl

Mobile Phase B: Buffer A + 2.0 mol/L NaCl Gradient: 120 min linear gradient from 0-100% B Flow-rate: 1.0 mL/min, Detection: UV @ 280 nm Sample : BSA (pI 4.7-4.9), 1 mg

Binding @ pH 4.5 (< pI of BSA)

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0

5

10

15

20

25

30

35

0 20 40 60 80 100 120

mAU

@ 2

80 n

m

Retention time (min)

pH 5.0

pH Dependence of Elution of Ovalbumin

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Binding @ pH 4.5 (pI 4.6)

Column: TOYOPEARL NH2-750F, 5 mm ID×5 cm (1 mL), Mobile Phase A: pH 4.5 & pH5: 30 mmol/L N-methyl piperazine;

pH 6.0: 20 mmol/L Bis-Tris pH 7.5: 20 mmol/L Tris-HCl

Mobile Phase B: Buffer A + 2.0 mol/L NaCl Gradient: 0-100% B, 120 min linear gradient Flow-rate: 1.0 mL/min, Detection: UV @ 280 nm Sample : Ovalbumin (pI 4.6), 1 mg

pH 4.5

pH 6.0

pH 7.5

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Recovery of Proteins

• Column: TOYOPEARL NH2-750F, 5 mm ID×5 cm (1 mL) • Mobile Phase A: 20 mmol/L Tris-HCl (pH 8.0) • Mobile Phase B: Buffer A + 2.0 mol/L NaCl (pH 8.0) • Gradient: 0-100% B, 20 min linear gradient, 100% B for 5 min • Flow-rate: 1.0 mL/min • Sample: 1 mg each

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Protein Recovery(%)

Ovalbumin 93Bovine serum albumin 97

Mab 94β-Lactoglobulin 95

Transferrin 100

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DBC of TOYOPEARL NH2-750F for different proteins

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TOYOPEARL NH2-750F has moderate capacity at the ip of a protein.

sample: IVIG, 150 cm/h10 mM bis-Tris 10 mM Tris/HCl 10 mM Tris/HClpH 6 pH 7 pH 8

c (NaCl) [mM] DBC c (NaCl) [mM] DBC c (NaCl) [mM] DBC0 1,4 0 7,6 0 47,8

50 1,9 50 6,3 50 18,1100 1,8 100 3,4 100 5,6200 1,6 200 2,1 200 2,2300 1,5 300 1,8 300 1,8500 1,5 500 1,5 500 1,5

sample: HSA, 150 cm/h10 mM bis-Tris 10 mM Tris/HCl 10 mM Tris/HClpH 6 pH 7 pH 8

c (NaCl) [mM] DBC c (NaCl) [mM] DBC c (NaCl) [mM] DBC0 23,5 0 32,2 0 44,6

50 8,4 50 15,8 50 35,9 100 4,8 100 9,2 100 30,7 200 2,4 200 4,1 200 18,9 300 2 300 2,4 300 11,2 500 1,8 500 2,5 500 5,4

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Separation of IgG1 and its Aggregates

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Column: TOYOPEARL NH2-750F (5 mm ID X 5 cm) Elution: 60-min linear gradient from 0 to 1 mol/L NaCl in 20 mmol/L Tris-HCl (pH 8.0) Flow rate: 1.0 mL/min, sample; Detection; UV (280 nm) Sample: mAb (IgG1, 0.5 mg) Fraction 2 includes dimer aggregates

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Purity Check by SEC

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Column: TSKgel® G3000SWXL, 7.8 mm I.D. X 30 cm Mobile Phase: 0.1 mol/L sodium phosphate containing 0.3 mol/L NaCl, pH 7.0 Flow rate: 1.0 mL/min Detection: UV @ 210 nm Sample: mAb (IgG1), original sample & fractions from TOYOPEARL NH2-750F

Original Sample

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mAb aggregate removal

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Column: TOYOPEARL NH2-750F (6 mm ID X 5 cm) Elution: 60 CV linear gradient from 0 to 0.35 mol/L NaCl in 10 mmol/L Tris-HCl, pH 7.0 Flow rate: 150 cm/h, Detection UV (280 nm) Sample: aggregated mAb (5 mg)

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DNA removal

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An aggregated mAb sample was spiked with eukaryotic DNA. DNA content was measured fluorometrically (Invitrogen Quant-iT dsDNA HS Assay Kit Q32854). Aggregate quantification by AUC @280 nm, TSKgel G3000SWxl, 1 ml/min, 100 mM NaP, 100 mM NaSulfate, pH 6.7

SEC - load SEC - product

Load product DNA content 130 µg/mL < 0.5 ng/mL

Aggregate content 16.0 % < LOD

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Benefits of AEX at the isoelectric point of a mAb

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•  No product deamidation at basic pH

•  Can prevent the need of a cation exchanger step

•  good platform applicability à e.g. compared to weak partitioning anion exchange chromatography

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Conclusions

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•  TOYOPEARL AF-rProtein-A HC 650F allows HCP reduction by 3 orders of magnitude

•  Leaching of TOYOPEARL AF-rProtein-A HC 650F is below 10 ng/ml, which corresponds to values smaller than 2.5 ppm.

•  TOYOPEARL NH2-750F allows aggregate removal at the isoelectric point of a mAb.

•  DNA can be reduced by more than 5.4 log with TOYOPEARL NH2-750F

•  We can now again start to think of 2-step chromatographic purification of mAbs

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Conclusions

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•  TOYOPEARL AF-rProtein-A HC 650F allows HCP reduction by 3 orders of magnitude

•  Leaching of TOYOPEARL AF-rProtein-A HC 650F is below 5 ng/ml, which corresponds to values smaller than 2.5 ppm.

•  TOYOPEARL NH2-750F allows aggregate removal at the isoelectric point of a mAb.

•  DNA can be reduced by more than 5.4 log with TOYOPEARL NH2-750F

•  We can now again start to think of 2-step chromatographic purification of mAbs

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TOYOPEARL NH2-750F was stored in 0.5 mol/L NaOH at 25°C.

Alkaline Stability

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TOYOPEARL NH2-750F

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Primary polyamine

Ligand Polyamine Particle size (mean) 45 µm Pore size (mean) > 100 nm Ion exchange capacity 0.10 ± 0.03 eq/L