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

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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


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

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


• 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

% o

f m

ax

. c

ap

ac

ity

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 ToyopearlAF-rProtein A-

650F

Resin B Resin C Resin D Resin E

DB

C (

mg

/ml)

80 % Capacity



23

0

10

20

30

40

50

60

70

80

90

100

Toyopearl AF-rProtein A


%

Recovery



24

88

88.5

89

89.5

90

90.5

TosohToyopearl

AF-rProteinA-650F


%

% Monomer

0

1

2

3

4

5

6

7

8

9

10

TosohToyopearl

AF-rProteinA-650F


%

% Dimer


0

0.5

1

1.5

2

2.5

3

Tosoh ToyopearlAF-rProtein A-

650F


Cost factor


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


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

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


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

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

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

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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

[email protected]

For all the latest news, events, applications and more,

connect with us

@TosohBio

or on CHROMlive.com

http://www.tosohbioscience.com/

mailto:[email protected]

Science

Dr. Elke Prohaska & Regina Römling BioInnovation Leader Summit Tosoh