BILS 2015 Tosoh Bioscience
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Transcript of BILS 2015 Tosoh Bioscience
Tosoh Bioscience
Making the Impossible Possible –
Chromatographic Solutions for Demanding
Separations in Downstream Processing
BioInnovation Leader Summit 2015 Judith Vajda, Regina Römling, Egbert Müller
Tosoh Bioscience impossible
Tosoh Bioscience 2
• 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
3
• 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
4
Primary Recovery
Protein A Capturing CEX AEX Final
Filtration
Primary Recovery
Protein A Capturing HIC IEX Final
Filtration
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Purification requirements
5
• 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
7
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?
11
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
13
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
14
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
15
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
16
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
17
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
18
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
19
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
20
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
23
• 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
24
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TOYOPEARL NH2-750F
25
Primary polyamine
Ligand Polyamine Particle size (mean) 45 µm Pore size (mean) > 100 nm Ion exchange capacity 0.10 ± 0.03 eq/L