TOSOH BIOSCIENCE
Improving Process Efficiency in
Biomanufacturing
Dr. Elke Prohaska & Regina Römling
BioInnovation Leader Summit 2013
TOSOH BIOSCIENCE
Outline
• Antibody Purification
• Save Water for Injection
• Mixed-Mode Resins
TOSOH BIOSCIENCE
Antibody Purification
Reduced Process Cycle Times and Costs in
Large and Small Scale mAb Purification -
Two Case Studies
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TOYOPEARL ® Process Media
• Rigid polymethacrylate matrix
• Broad range of particle & pore sizes
• Enable high flow rates in large columns
• SEC, IEX, HIC & Affinity media
• NEW: Mixed-Mode Medium
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Typical Antibody Purification
Polishing II
Virus
Filtration
Polishing I
acidic pH
Capture
Formulation
Protein A Affinity Chromatography
CEX
AEX
AEX
CEX
HIC
AEX
MX
AEX
TOSOH BIOSCIENCE
High Capacity Toyopearl Resins
Protein A Affinity Resin: • TOYOPEARL AF-rProtein A-650F
Ion Exchange Resins:
• TOYOPEARL GigaCap ® series
• TOYOPEARL GigaCap S-650M
• TOYOPEARL GigaCap CM-650M
• TOYOPEARL GigaCap Q-650M
Hydrophobic Interaction Resins:
• TOYOPEARL HIC-600 series
• TOYOPEARL PPG-600M
• TOYOPEARL Butyl-600M
• TOYOPEARL Phenyl-600M
New Multimodal CEX Resin: • TOYOPEARL MX-Trp-650M
> 90 g/L DBC for IgG; Applicable for aggregate removal
TOSOH BIOSCIENCE
TOYOPEARL AF-rProtein A-650F
Key features:
• High IgG binding at high flow/short residence time
• Efficient capturing out of high titer feedstocks
• Chemical (caustic/acidic), thermal, mechanical stable
• Rigid polymer matrix
• High purity of antibody
• Low protein A leakage
• Fast kinetics
• Attractive price
Purification of
• large amounts of antibody
• in shorter time
• at lower costs
Breakthrough curves for hIgG (10 mg/mL)
Column : 5 mm ID x 5 cm (1mL); Mobile phase: 20 mM sodium
phosphate (pH 7.2) , 150 mM NaCl
TOSOH BIOSCIENCE GmbH
Case study I - InVivo
• InVivo: • Non-GMP contract manufacturer for monoclonal antibodies and
other recombinant proteins for R&D, diagnostic and preclinical
samples
• 1600 different mAB hybridomas, 10 mg – 500 g scale, ca. 10 kg
purified antibodies, vaious species, various sub classes (IgG1 –
IgG 4, IgM, heterohybridomas)
• 12 fermentation units 2 to 100 L
• Target: deliver high quality at moderate/low costs
• Requirements for the mAb capture resin: • Low process costs
• Excellent cleaning properties
8
• Sample: 10.0 Liter cell culture supernatant (0.40 mg/mL)
• Total yield mAb: 4 g
• Average binding capacity of Protein A:
approx. 10 mg/mL
• CV: 400 mL, Flow rate: 60 mL/min
• Equilibration 1 CV
• Residence time: 6 min
• Wash 5 CV
• Total elution 5 CV
• Re-equilibration 2 CV
• Sample time: approx. 170 min
Established IgG-Purification Scheme
Comparison of Resins
Tosoh Toyopearl AF-rProtein A-650F Company A Resin A Company M Resin M Invivo‘s requirements: • Approx. 100 runs -> 100 CIPs • Only small loss of dynamic binding capacity • Very good purity (95%) after one step purification
According to vendor
AF-rProtein A-650F
Resin A Resin M
Binding capacity Human IgG
≥ 30mg/ml ≥ 30mg/ml ≥ 50mg/ml
Pressure stability 3 bar 100 bar 10 bar
Performance
3 columns of same size
3 different resins
2 cell culture supernatants of different but well known antibodies
All are mouse IgG2a
Eluate
Neutralization
Yield check
Desalting
Concentration
Sterile Filtration
QC
Yield check
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Test with two Antibodies
12
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Antikörper A Antikörper B
Recovery
TOYOPEARL AF-rProtein A Resin 1 Resin 2
TOSOH BIOSCIENCE GmbH
Most Important – Stability towards NaOH CIP
13
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
1 10 80 160
CIP Zyklen
DBC
After 80 CIP-cycles with 0,5 M NaOH > 80 % of initial binding capacity!
CIP : 2 CV wash buffer 0.2M NaPP, 10 CV 0,5M NaOH (t ≥ 10 min), 10 CV wash buffer
Purity [%]
50
60
70
80
90
100
Pu
rity
[%
]
50
60
70
80
90
100
1 10 100 180
Pu
rity
[%
]
Purity after x CIPs
AK 1 AK 2 AK 3
AF-rProtein A-650F
Competitive Resin A
Competitive Resin M
Evaluation - DBC
Costs [based on €/L]:
(August 2011)
DBC • AF-rProteinA-650F
DBC • Resin M
DBC • Resin A
AF-rProteinA-650F
Resin M Resin A
1 X 1.46 X 1.86 X
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Results – Case Study I
TOYOPEARL AF-rProtein A-650F advantages:
• High mAb concentration in product fraction
• Reasonable price
• Intensive CIP possible
• Good DBC with higher flow rates
TOSOH BIOSCIENCE GmbH
• Target
Determine ideal conditions for mAB purification
• Comparison of 6 Protein A resins
• „Dirty“ Feedstock (20 % Aggregate)
max. Recovery = 80 % Monomer
17
Case Study II – Capacity Screening Results
for Large Scale Purification
TOSOH BIOSCIENCE GmbH
Pre-Screening – Define Main Parameters
18
0
5
10
15
20
25
30
35
40
45
50
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
80
% o
f m
ax
. c
ap
ac
ity
Residence time (min)
Toyopearl AF-rProtein A-650F B C D E F
TOSOH BIOSCIENCE GmbH
Elution Study TOYOPEARL AF-rProtein A (1)
• Conditions: • Residence time: 3.5 min (best productivity for this process)
• Column Volume: 7.98 mL
• Buffer Conditions: • Equilibration/Wash: PBS pH 7,4
• Elution: variable
• CIP: 0,1 M NaOH
• Reequilibration: PBS pH 7,4
• Storage: 20 % EtOH
19
TOSOH BIOSCIENCE GmbH
Elution Study TOYOPEARL AF-rProtein A (2)
20
Elution Buffer pH
& Salt
3,0 3,5 3,5 3,5 3,5 3,5 3,7 3,7 3,7 3,9 3,9
0,1M Gly 0,1M Cit 0,1M Ace 0,1M Gly
0,1M Gly
+ 0,1M
Arg
0,1M Gly
+ 0,5M
Arg
0,1M Gly
0,1M Gly
+ 0,1M
Arg
0,1M Gly
+ 0,5M
Arg
0,1M Gly
0,1M Gly
+ 0,1M
Arg
Monomer (%) 77,1 70,5 79,0 78,2 82,5 81,0 86,2 87,9 84,4 88,2 93,7
Dimer (%) 18,0 16,2 17,2 18,3 15,3 15,1 11,9 11,1 13,7 10,0 6,0
Rest (%) 4,9 13,3 3,8 3,5 2,2 3,9 0,2 1 1,9 1,8 0,3
Recovery (%) 73,4 66,4 71,1 75,5 76,0 79,3 74,7 76,1 76,7 63,3 55,9
Elution Buffer pH
& Salt
3,9 3,9 4,0 4,0 4,0 4,0 4,0 4,0 4,0 4,0 4,0
0,1M Gly
+ 0,5M
Arg
0,1M Gly
+ 0,5M
Arg
0,1M Cit 0,1M Cit +
0,1M Arg
0,1M Cit +
0,5M Arg
0,1M Cit +
1M Arg 0,1M Ace
0,1M Ace
+ 0,1M
Arg
0,1M Ace
+ 0,5M
Arg
0,1M Ace
+ 1 M Arg
0,1M Ace
+ 0,5M
NaCl
Monomer (%) 89,5 89,5 91,4 93,4 91,1 83,6 93,2 93,0 89,7 85,6 95,0
Dimer (%) 9,6 9,6 7,6 4,9 8,2 12,5 6,0 5,4 8,6 12,8 4,4
Rest (%) 0,9 0,9 1 1.7 0,7 3,9 0,8 1,6 1,7 1,6 0,6
Recovery (%) 75,8 75,8 33,9 50,6 73,5 83,4 53,0 61,2 74,9 80,9 46,4
TOSOH BIOSCIENCE GmbH
Protocoll for TOYOPEARL AF-rProtein A
21
Buffer equilibration 6 CV PBS pH 7.4
Buffer wash 6 CV PBS pH 7.4
Buffer elution 8 CV 0.1 M acetate + 0.5 M arginine pH 4.0
Buffer CIP 6 CV 0.1 M NaOH
Buffer reequilibration 10 CV PBS pH 7.4
Buffer storage 6 CV EtOH 20 %
TOSOH BIOSCIENCE GmbH
Comparison using Optimized Method
22
0
5
10
15
20
25
30
35
40
45
50
Tosoh ToyopearlAF-rProtein A-
650F
Resin B Resin C Resin D Resin E
DB
C (
mg
/ml)
80 % Capacity
TOSOH BIOSCIENCE GmbH
Comparison using Optimized Method
23
0
10
20
30
40
50
60
70
80
90
100
Toyopearl AF-rProtein A
Resin B Resin C Resin D Resin E
%
Recovery
TOSOH BIOSCIENCE GmbH
Comparison using Optimized Method
24
88
88.5
89
89.5
90
90.5
TosohToyopearl
AF-rProteinA-650F
Resin B Resin C Resin D Resin E
%
% Monomer
0
1
2
3
4
5
6
7
8
9
10
TosohToyopearl
AF-rProteinA-650F
Resin B Resin C Resin D Resin E
%
% Dimer
TOSOH BIOSCIENCE GmbH
0
0.5
1
1.5
2
2.5
3
Tosoh ToyopearlAF-rProtein A-
650F
Resin B Resin C Resin D Resin E
Cost factor
Comparison using Optimized Method
25
Costs calculated for 3 runs
(Incl. chromatography resins and buffer consumption)
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Cost Comparison
Includes costs of required resin and costs of buffers
Name Resin Toyopearl AF-
rProtein A-650F
Resin B Resin C Resin D Resin E
80 % Capacity
(mg/ml) @3.5
min RT)
44 33 31 22 22
Recovery (%) 74.9 79.8 71.4 77.0 76.5
Monomer (%) 89.7 90.1 89.9 89.5 88.8
Dimer (%) 8.6 9.3 8.7 7.0 5.7
Cost factor (%) 100 137 141 221 284
TOSOH BIOSCIENCE GmbH
Resin Stability & Process Costs
0
5
10
15
20
25
30
35
40
45
50
mg
/ml
80 % DBC
0
0.5
1
1.5
2
2.5
3
3.5
€/g
mA
B
Material/Prozess - Kosten
0.1 M NaOH CIP Cycles
200
300 60 160
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Summary of Results
• Reduced cycle time √
• Reduced costs small scale √
• Reduced costs large scale √
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Outline
• Antibody Purification
• Save Water for Injection
• Mixed-Mode Resins
TOSOH BIOSCIENCE GmbH
Save Water for Injection with
TOYOPEARL® Resins
TOSOH BIOSCIENCE GmbH
Rising Titers – Limited DSP Facilities
• Upstream development achieve high protein titers of up 10 g/L
produced in existing bioreactor equipment.
• Challenge for DSP to handle this increased biomass
• Critical issue: Titer is proportional to the volume of WFI (water
for injection) needed for DSP.
If a bioreactor in a given process
is producing a target at a 1 g/L titer
and downstream needs 10,000 L of
WFI to process it, then the same
bioreactor with an improved titer of
3 g/L will require 30,000 L of WFI to
process the increased protein mass.
In some cases, 25,000 L of feed-stock
may require 125,000 L+ of WFI per
process cycle (incl. affinity, cation,
anion chromatography steps & UF/DF)
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Options to Overcome DSP Bottleneck
• Increase numbers of cycles
• Increase existing tanks/bags in size or number
• Increase columns size or number
• Use resin with higher capacity versions
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High Capacity & Fast Kinetics
• High binding capacity – more feedstock to be processed with
existing column size
• Smaller resin/column volume - reduced buffer/WFI consumption
for all steps (equilibration, wash, CIP, later DSP steps).
• All benefits undone if elution peak tails!
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Effect of ideal DBC & Kinetics
38 mL elution volume for the
agarose resin
8 mL elution volume for
TOYOPEARL GigaCap® S
FACTOR >4 !
Lower pool volume /higher
product pool titer only based
on better kinetics.
TOSOH BIOSCIENCE GmbH
Summary
High DBC and good elution dynamics of
TOYOPEARL GigaCap ® IEX resins result in
significantly less tailing in the elution process with
up to 75% less buffer volume required for the
target fractions, when compared to commercial
agarose based resins:
Keep the hardware and resin bed volume constant
for increased biomass load and even save up to
75% WFI for elution.
Additional cost saving by reduced size of bags/
columns/filters applied for further purification of the
target.
TOSOH BIOSCIENCE GmbH
What about other Modes?
NEW mixed-mode resin TOYOPEARL MX-Trp-650M offers
similar advantages
• Multimodal cation exchanger
• Multiplies binding capacity of current mixed-mode resins
• Tolerates high ionic strength –> no dilution
• CIP resistant
• Fast kinetics – small elution volume
• Suitable for aggregate removal
Resin and buffer consumption ▼ + Columns/buffer tanks etc. ▼
= lower cost of goods/g purified target ▼
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Why Use Mixed Mode?
•Direct load of high conductivity feedstocks
•Additional chromatographic mode with unique
selectivity that combines hydrophobic and ion
exchange characteristics
•Selectivity can be modulated by salt and pH variation
•Suitable for aggregate removal in
intermediate/polishing steps
37
TOSOH BIOSCIENCE GmbH
TOYOPEARL MX-Trp-650M
TOSOH BIOSCIENCE GmbH 39
Sharper Peaks – Less Peak Tailing
Resins: TOYOPEARL MX-Trp-650M, Brand M;
Column size: 7.5 mm ID x 7.5 cm
Mobile phase: Buffer A: 20 mmol/L phosphate (pH 7.0); Buffer B: 20 mmol/L phosphate
+ 1.0 mol/L NaCl (pH 7.0)
Gradient: 30 min linear from buffer A to buffer B
Flow rate: 1.0 mL/min
Detection: UV @ 280nm
Sample: Trypsinogen (6.6 mg/mL); cytochrome C (3.6 mg/mL); lysozyme (6.6 mg/mL);
Sample vol.: 25 μL
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Low Pool Volume Elution Peak
Column: TOYOPEARL MX-Trp-650M (6 mm ID x 4 cm)
Binding buffer; 0.05 mol/L acetate buffer (pH 4.7) + 0.1 mol/L NaCl
Elution buffer: 0.1 mol/L Tris-HCl buffer (pH 8.5) + 0.3 mol/L NaCl
Adsorption flow rate: 1.0 mL/min (212 cm/h)
Elution flow rate: 2.0 mL/min (started at 124 min)
Detection: UV @ 280 nm
Sample: CHO cell culture media, monoclonal antibody (1 mg/mL) diluted with binding buffer
Tosoh Bioscience 41
Ideal Pressure Flow Properties
Back pressure of TOYOPEARL MX-Trp-650M column is less than half of other
commercial multimodal CEX resins
Resin: TOYOPEARL MX-Trp-650M (red) Agarose based multimodal CEX gel (blue)
Column size: 22 mm ID x 20 cm
Eluent: distilled water
Tosoh Bioscience 42
High DBC @ High Conductivity
Resin Mean
Particle Size
DBC
@12 mS/cm
Recovery
@12 mS/cm
DBC
@17 mS/cm
Recovery
@17 mS/cm
TOYOPEARL MX-Trp 75 µm 95 g/L 97 % 48 g/L 96 %
Brand M (Agarose) 75 µm 14 g/L 86 % 11 g/L 85 %
Toyopearl MX-Trp-650M or Brand M agarose based multimodal CEX
Column size: 6 mm ID x 4 cm
Binding Buffer: 50 mmol/L acetate buffer (pH 4.7) + 100 mmol/L NaCl (conductivity 12 mS/cm) or
50 mmol/L acetate buffer (pH 4.7) + 150 mmol/L NaCl (conductivity 17 mS/cm)
Flow rate: 212 cm/h; Detection: UV @ 280 nm;
Sample: polyclonal IgG
DBC measured at 10 % breakthrough
Recovery and binding capacity of TOYOPEARL MX-Trp-650M at high conductivity is
much higher than those of other commercial multimodal CEX resins
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Aggregate Removal – mAb Feedstock
43
SEC analysis on standard TSKgel G3000SWXL revealed aggregate content of ~17%
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Aggregate Removal – Mixed-Mode Run
44
TOYOPEARLMX-Trp-650M
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Aggregate Removal – Result
45
SEC analysis on standard TSKgel G3000SWXL
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Aggregates – Fast SEC on UHPLC
Mobile phase: 0.1 M NaP, 0.1 M NaSulfate,
pH 6,7
Flow rates: 0.7mL/min -: 0.35 mL/min
Injection vol. : 5 μL
Temperature: 25℃
Detection: UV @ 280 nm
Samples: aggregated mAb (1 mg /mL)
Column: TSKgel SuperSW mAb HTP,
4.6 x 150 mm
TSKgel® SuperSW mAb HTP combined with UHPLC System keeps
resolution high at less than half of the standard analysis time!
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Separation of mAb/mAb Fragments
47
SEC analysis on standard TSKgel G3000SWXL
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Conclusion
Modern resins that combine high DBC,
fast kinetics and high flow rates
help to
• use current hardware with rising
upstream titers
• save buffer volume
• increase process throughput
and save time and money in biologics production.
48
TOSOH BIOSCIENCE
The Antibody Toolbox
• The antibody purification toolbox:
• TOYOPEARL AF-rProtein A – alkaline stable & high capacity at high flow
• TOYOPEARL GigaCap – High capacity IEX, low elution volume at high flow
• TOYOPEARL HIC-600M – high capacity due to pore size optimization for IgG
• TOYOPEARL MX-Trp – new selectivity and high DBC for mAb polishing
• The analytical antibody toolbox for QC:
• TSKgel SW – the industry‘s standard for aggregate analysis
• TSKgel STAT – fast IEC analysis of charge isoforms
• TSKgel Amide-80 – HILIC for glycosylation analysis
• TSKgel Protein C4-300 – reversed phase for fragment analysis
• Fast method development with automated screening using
NEW ToyoScreen® RoboColumns ®
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Summary
• Modern chromatography media for all
purification steps facilitate processing of
increased upstream titers.
• High binding capacity, high flow rates, and
small elution volumes keep volumes/hardware
dimensions low and DSP throughput high.
• Analytical tools need to
keep the pace with
production progresses
TOSOH BIOSCIENCE
Contact Details
Tosoh Bioscience GmbH
Zettachring 6 – 70567 Stuttgart
www.tosohbioscience.com
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