Continuous Antibody Capture with Protein A Countercurrent Tangential

Chromatography: A New Column-Free Approach for Antibody

Purification

Andrew L. Zydney Department Head and Walter L. Robb Family Chair

Department of Chemical Engineering The Pennsylvania State University

Presented at the ECI Conference on Integrated Continuous Biomanufacturing

Castelldefels, Spain, October 21, 2013

• Significant potential opportunities – Reduced capital costs / facility requirements – Higher productivity – Easier scale-up

• Major technology developments in place – Perfusion bioreactors – In-line filters

• Critical challenge is chromatography

Continuous Bioprocessing

• Multi-column periodic counter-current chromatography (PCC) – GE Healthcare

• Simulated moving bed chromatography (SMB) – Semba, Tarpon, Contichrom

• Sequential multi-column chromatography (SMCC) – Novasep

• These approaches typically do not provide truly steady-state operation, potentially leading to variability in product quality

Chromatography Options

Example: SMB

New Developments in Simulated Moving Bed Chromatography Seidel-Morgenstern, Kessler, and Kaspereit Chemical Engineering Technology, 31: 826 (2008)

• Develop and demonstrate a new technology that can provide truly continuous protein purification using available chromatography resins, e.g., Protein A

• Design criteria: – Comparable yield and purity to columns – High productivity (10x packed columns) – Single use capability (no stainless steel)

Objectives

Countercurrent Tangential Chromatography - CTC

Chromatographic resin (beads) flows as a slurry through a series of static mixers and hollow fiber membrane modules All operations (binding, washing, elution,

stripping, equilibration) performed directly on the slurry Countercurrent staging used to reduce buffer

and resin requirements, increase product yield and purity

Continuous CTC System

Binding Washing Elution Stripping Regnera-tion

Slurry Tank

Feed Tank

Waste

Waste

Waste Waste

Product Tank

True moving bed Conveyor like process Resin slurry moves counter-currently to buffer in each step

Chromatographic “Stage”

Provides residence time needed for equilibration in binding and elution steps

Excellent radial mixing with minimal pressure drop

Provides complete separation between resin particles and fluid phase

High single pass conversion with low pressure losses

+

Koflo static mixer

Spectrum hollow fiber module

Centrate Feed

Wash 1 Buffer

Wash 2 Buffer

Elution Buffer

Stripping Buffer

Equilibra-tion

Buffer

Continuous CTC System

Resin Tank

UF Step

Binding

Wash 1

Wash 2

Elution

Stripping

Equilibration

Binding Permeate

Wash 1 Permeate

Product Tank

UF Permeate

Wash 2 Permeate

Stripping Permeate

Equilibra-tion

Permeate

Countercurrent Staging - Elution

1st stage

2nd stage

Washing

pH 3 Elution Buffer

Purified mAb

Resin Slurry from Wash

Resin Slurry to Strip

Stage 1 Stage 2

1st stage

2nd stage

Washing

pH 3 Elution Buffer

Purified mAb

Resin Slurry from Wash

Resin Slurry to Strip

Stage 1 Stage 2

Countercurrent Staging - Elution

1st stage

2nd stage

Washing

pH 3 Elution Buffer

Purified mAb

Resin Slurry from Wash

Resin Slurry to Strip

Stage 1 Stage 2

Countercurrent Staging - Elution

1st stage

2nd stage

Washing

pH 3 Elution Buffer

Purified mAb

Resin Slurry from Wash

Resin Slurry to Strip

Stage 1 Stage 2

Countercurrent Staging - Elution

1st stage

2nd stage

Washing

pH 3 Elution Buffer

Purified mAb

Resin Slurry from Wash

Resin Slurry to Strip

Stage 1 Stage 2

Countercurrent Staging - Elution

Tangential flow filter

Static mixer

Static mixer

Tangential flow filter

Tangential flow filter

Static mixer

Concentrated slurry with

bound product

Concentrated resin slurry

Elution Buffer

Product

P

P

R

P R

R

Example: 3-Stage Elution Step

Number of stages

Experimental yield

Theoretical yield

1 78 ± 2% 77%

2 94 ± 2% 94%

3 98 ± 1% 98%

qp = permeate flow rate qr = retentate flow rate n = number of stages

Effect of Staging – Elution Step

Results for qp / qr = 0.75

From Shinkazh et al., Biotech. Bioeng, 108: 582 (2011)

Experimental System

• Clarified cell culture fluid (Fujifilm Diosynth) – Monoclonal antibody product

• POROS® MabCapture A resin – Life Technologies – 45 µm diameter particles, Protein A ligand

• MidiCros® hollow fiber modules - Spectrum Lab – 0.5 µm PES membranes, 1 mm ID, 200 cm2 area

• Static mixers – Koflo Corportation – 29 cm length, 1 cm ID

Critical Filtrate Flux

170

200

230

260

290

0.6

0.8

1.0

1.2

1.4

0 500 1000 1500 2000

Filtrate Flux, Jv (L m

-2 hr -1)

Tran

smem

bran

e Pr

essu

re, T

MP

(psi

)

Time, t (s)

TMP

Flux

Feed: 10% slurry, 100 mL/min

Critical Filtrate Flux

170

200

230

260

290

0.6

0.8

1.0

1.2

1.4

0 500 1000 1500 2000

Filtrate Flux, Jv (L m

-2 hr -1)

Tran

smem

bran

e Pr

essu

re, T

MP

(psi

)

Time, t (s)

TMP

Flux

Critical Flux

Feed: 10% slurry, 100 mL/min • Critical flux corresponds to 80% conversion using 10% slurry

Critical Filtrate Flux

170

200

230

260

290

0.6

0.8

1.0

1.2

1.4

0 500 1000 1500 2000

Filtrate Flux, Jv (L m

-2 hr -1)

Tran

smem

bran

e Pr

essu

re, T

MP

(psi

)

Time, t (s)

TMP

Flux

Critical Flux

Operating Flux

Feed: 10% slurry, 100 mL/min • Critical flux corresponds to 80% conversion using 10% slurry

• System design: – 7.5% slurry – 75% conversion

• Extra safety limit enables stable operation for long times

CTC Process using Protein A

Operation Numbe

r of stages

Buffer pH Mixed pH

Binding 2 -- 7.4 7.6

Wash 1 3 20 mM Na2HPO4 + 0.5 M NaCl 7.1 7.5

Wash 2 3 20 mM Na2HPO4 7.2 7.0

Elution 3 40 mM Citrate 3.2 3.3

Strip 2 10 mM HCl + 0.1 M NaCl 2.0 2.5

Equilibration 2 20 mM Na2HPO4 8.1 7.0

Multiple Runs

Run mAb (g/L)

mAb Load

Run Time

Feed Flow Rate

(L/hr)

mAb Load per

Resin

1 – feasibility 1.2 16 g 3 hr 4.5 190 g/L

2 – long time 0.72 8 g 24 hr 0.45 470 g/L

3 – high titer 4.5 8 g 4 hr 0.45 230 g/L

Run 1 - Pressure Profiles

Elapsed Time, t (hr)

Pres

sure

, P (p

sig)

0

2

4

6

8

10

12

0 0.5 1.0 1.5 2.0 2.5

• Stable operation

• Pressure <10 psi

• Laminar flow

• All plastic tubing and connectors

Run 1- mAb Purification

Elution Pure mAb CCCF

SEC Profiles

Elution Time, t (min)

Abs

orba

nce

• >95% yield • >98% purity • Productivity of

63 g mAb/L resin/hr (10x packed column)

• No detectable protein aggregates

• No detectable changes in resin

Run 1 - mAb Purification

Sample Host Cell Protein (ppm)

Clarified Harvest 675,000

CCTC System 1,200

Packed Column 2,800

• Host cell protein measured relative to mAb via ELISA • HCP level in CCTC system 2x lower than packed column • Yield >95%, purity >98% • Similar levels of high MW to purified (reference) mAb

Run 2 – Product Profile

0

0.2

0.4

0.6

0.8

1

0 2 4 6 8 10 12

UV

- E

lutio

n

Time (hours)

• Steady-state with respect to product concentration and impurity profile

• Long time operation possible

• For t > 12 hr hollow fiber modules had to be replaced due to bacterial growth

Run 2 – HCP levels

• HCP level remains constant throughout 24 hr run

• >95% purity • Productivity of

19 g mAb/L resin/hr (reduced due to low titer feed)

Run 3 - mAb Purification

Sample Host Cell Protein (ppm)

2 hr 310

3 hr 345

4 hr 382

• Very low HCP level due to use of high titer feed (4.5 g/L) spiked with purified mAb

• >99% purity • Productivity of 52

g mAb/ L resin / hr • 2.6 cycles / hr for

resin • HCP measured via ELISA relative to mAb

Advantages of CCTC System • Continuous operation with high productivity

– All resin used at all times – Steady-state operation with respect to product

concentration and impurity profiles

• No columns / packing – Reduced labor costs and validation – Greater flexibility in multi-product facilities

• Disposable flow path if desired – Potential for single-use systems – Ideal for production of clinical batches

Future Opportunities • Use of smaller resin particles

– Much better mass transfer less residence time needed in binding and elution steps

– Lower hold-up volume greater productivity – No issues with pressure drop for slurry flow

• Direct integration with perfusion bioreactor – Opportunity for continuous steady-state processing – Dramatic improvements in overall productivity

Summary • Countercurrent tangential chromatography

(CCTC) for mAb purification – Continuous and steady-state operation demonstrated

for 24 hr – Purity and yield comparable to packed column – Countercurrent staging reduces resin requirements

while increasing product yield and purity – Low pressure operation opportunities for

disposable single-use flow path – Modular design for enhanced flexibility

Acknowledgements • Oleg Shinkazh

– Founder and President, Chromatan • Boris Napadensky

– VP of Engineering, Chromatan

• Achyuta Teella – Senior Scientist at Chromatan, Post-doc at Penn State

• Travis Tran – Associate Scientist, Chromatan

• Gary Brookhart – Senior Research Scientist, Fujifilm Diosynth

Funding / Support