BILS 2015 Tosoh Bioscience "Making the Impossible Possible – Chromatographic Solutions for Demanding Separations in Downstream Processing" Judith Vajda, Regina Römling and Egbert Müller

1. 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

2. 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

3. Tosoh Bioscience
Outline
3
• mAb platform approach
• Requirements and solutions for mAb
capturing in a 2-step scenario
• mAb Polishing within one
chromatographic step

4. Tosoh Bioscience
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

5. Tosoh Bioscience
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.

6. Tosoh Bioscience
Capturing - Protein A chromatography
6
• High capacity allows product concentration
• Extensive HCP removal
• Low protein A leakage
Capturing with HC Protein A

7. Tosoh Bioscience
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

8. Tosoh Bioscience
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
LOGunitsofCHOPreduction
no. of experiment
TP AF rProteinA HC 650F (Citrate) TP AF rProteinA HC 650F (Acetate)

9. Tosoh Bioscience
Titer dependency – CHOP content
High titer feedstreams improve CHOP clearance of protein A
chromatography!
% Spiking (v/v) with 10 x concentrated CCF

10. Tosoh Bioscience
Protein A leakage
Protein A leakage of TOYOPEARL AF-rProtein A-HC 650F is
smaller than 10 ng/ml.
Design-Expert® Software
ProteinA leaching
10
0
X1 = A: pH
X2 = B: load
Actual Factors
C: titer = 4.75
D: 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
40.00
ProteinA leaching
elution pH
columnload[mg/mL]
2
2
468
40.0
35.0
30.0
25.0
20.0
2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25
68 24
Design-Expert® Software
ProteinA leaching
10
0
X1 = A: pH
X2 = B: load
Actual Factors
C: titer = 4.75
D: spiking = 15.00
2.25 2.50 2.75
20.00
25.00
30.00
35.00
40.00
Pro
columnload[mg/mL]
46
8
2
gn-Expert® Software
inA leaching
0
A: pH
B: load
al Factors
er = 4.75
iking = 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
40.00
ProteinA leaching
elution pH
columnload[mg/mL]
246
8
40.0
35.0
30.0
25.0
20.0
2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25
6
8
24
Design-Expert® Software
ProteinA leaching
10
0
X1 = A: pH
X2 = B: load
Actual Factors
C: titer = 4.75
D: 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
40.00
ProteinA leaching
elution pH
columnload[mg/mL]
246
8
Citrate buffer Acetate buffer

11. Tosoh Bioscience
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?

12. Tosoh Bioscience 12
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
12
NH2-750F shows
higher retention than
other AEX media

13. Tosoh Bioscience
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)

14. Tosoh Bioscience
0
5
10
15
20
25
30
35
0 20 40 60 80 100 120
mAU@280nm
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

15. Tosoh Bioscience
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 93
Bovine serum albumin 97
Mab 94
β-Lactoglobulin 95
Transferrin 100

16. Tosoh Bioscience
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/h
10 mM bis-Tris 10 mM Tris/HCl 10 mM Tris/HCl
pH 6 pH 7 pH 8
c (NaCl) [mM] DBC c (NaCl) [mM] DBC c (NaCl) [mM] DBC
0 1,4 0 7,6 0 47,8
50 1,9 50 6,3 50 18,1
100 1,8 100 3,4 100 5,6
200 1,6 200 2,1 200 2,2
300 1,5 300 1,8 300 1,8
500 1,5 500 1,5 500 1,5
sample: HSA, 150 cm/h
10 mM bis-Tris 10 mM Tris/HCl 10 mM Tris/HCl
pH 6 pH 7 pH 8
c (NaCl) [mM] DBC c (NaCl) [mM] DBC c (NaCl) [mM] DBC
0 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

17. Tosoh Bioscience
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

18. Tosoh Bioscience
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

19. Tosoh Bioscience
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)

20. Tosoh Bioscience
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

21. Tosoh Bioscience
Benefits of AEX at the isoelectric point of a mAb
21
• 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

22. Tosoh Bioscience
Conclusions
22
• 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

23. Tosoh Bioscience
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

24. Tosoh Bioscience
TOYOPEARL NH2-750F was stored in 0.5 mol/L NaOH at 25°C.
Alkaline Stability
24

25. Tosoh Bioscience
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