Dr. Elke Prohaska & Regina Römling BioInnovation Leader Summit Tosoh

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

TOSOH BIOSCIENCE
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

TOSOH BIOSCIENCE
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

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

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
Purity [%]
50
60
70
80
90
100
1
10
100
180
Purity [%]
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

TOSOH BIOSCIENCE
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

•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

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 % of max. capacity
Residence time (min)
Toyopearl AF-rProtein A-650F
B
C
D
E
F

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

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

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 %

Comparison using Optimized Method
22
0
5
10
15
20
25
30
35
40
45
50
Tosoh Toyopearl
AF-rProtein A-
650F
Resin B
Resin C
Resin D
Resin E
DBC (mg/ml)
80 % Capacity

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

24
88
88.5
89
89.5
90
90.5
Tosoh
Toyopearl
AF-rProtein
A-650F
Resin B
Resin C
Resin D
Resin E
%
% Monomer
0
1
2
3
4
5
6
7
8
9
10
Tosoh
Toyopearl
AF-rProtein
A-650F
Resin B
Resin C
Resin D
Resin E
%
% Dimer

0
0.5
1
1.5
2
2.5
3
Tosoh Toyopearl
AF-rProtein A-
650F
Resin B
Resin C
Resin D
Resin E
Cost factor
25
Costs calculated for 3 runs (Incl. chromatography resins and buffer consumption)

TOSOH BIOSCIENCE
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

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 mAB
Material/Prozess - Kosten
0.1 M NaOH CIP Cycles
200
300
60
160

TOSOH BIOSCIENCE
Summary of Results
•Reduced cycle time √
•Reduced costs small scale √
•Reduced costs large scale √

Save Water for Injection with TOYOPEARL® Resins

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)

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

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!

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.

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.

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 ▼

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

TOYOPEARL MX-Trp-650M

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

Tosoh Bioscience
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

Tosoh Bioscience
Aggregate Removal – mAb Feedstock
43
SEC analysis on standard TSKgel G3000SWXL revealed aggregate content of ~17%

Tosoh Bioscience
Aggregate Removal – Mixed-Mode Run
44
TOYOPEARLMX-Trp-650M

Tosoh Bioscience
Aggregate Removal – Result
45
SEC analysis on standard TSKgel G3000SWXL

TOSOH BIOSCIENCE
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!

TOSOH BIOSCIENCE
Separation of mAb/mAb Fragments
47
SEC analysis on standard TSKgel G3000SWXL

Tosoh Bioscience
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 ®

TOSOH BIOSCIENCE
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 info.tbg@tosoh.com For all the latest news, events, applications and more, connect with us @TosohBio or on CHROMlive.com

Dr. Elke Prohaska & Regina Römling BioInnovation Leader Summit Tosoh

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