Platform downstream processes in the age of continuous chromatography: A case study

Mark Brower

BioProcess Technology & Expression Bioprocess Development

Kenilworth, NJ

Integrated Continuous Biomanufacturing Castelldefels, Spain 20-24 October 2013

Batch Stainless / Single Use

Batch Stainless

Continuous Single Use Enabled

PROCESS INTENSIFICATION

Next Generation

Transition to Future Concepts

To meet increasing global demands requires…

6H

18H

24H

30H

36H

42H

48H

54H

60H

12H

Primary Recovery (Centrifugation / MF + DF)

Bulk Purification Protein A Chromatography

Viral Inactivation (Low pH Hold)

DNA / HCP / Viral Adsorption Anion Exchange Chromatography

Variant and Aggregate Clearance Cation Exchange Chromatography

Viral Filtration Nanofiltration

Concentration / Buffer Exchange Microfiltration / Diafiltration

Bioburden Reduction Sterile Filtration

mAb Downstream Purification B

ulk

Form

ulat

ion

Fine

• Increased flexibility

• Reduced footprint

• Reduced capital spend

• Better resource utilization

Continuous Processing Vision - 2,000L SUB*

Overall DSP Time Cycle is Dictated by the Longest Step Other Steps are Lengthened to Compensate

S U B*

Depth /BRF

Filtration

Surge Bag

BioSMB Protein A

Single-Use Centrifugation

Surge Bag

p p

BRF p p

BRF p p

Viral Filtration

Surge Bag

Surge Bag

Formulation: BRF/DiaF

Continuous UF Anion

Exchange Membrane

p

p

Polishing Step

Continuous Viral Inactivation

A E X M

BRF

Surge Bag

*Single-Use Bioreactor

Hour 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 31 32 33

kSep 9:00 7:15

DF/SF 9:30 12:00

SMB 11:00 12:15

VI 11:40 1:30

AEX 6:00 4:00

MMC 7:30 4:15

UF 9:00 5:00

SUB

Harvest Bag

DF / BRF

SU Centrifuge SMB Protein A

Viral Inactivation

AEX Membrane

Mixed Mode

SPTFF

Continuous Processing Case Study mAb 1 - Non-platform

MCC for Bind & Elute Applications C1

C5

C3 C

7

Switch Time

• Methods based on batch process

• Loading, washing, elution, CIP carried out simultaneously

• Flexibility in loading zone

CEX CMCC Load Zone Design

2 methods designed to maximize time in the elution zone Wash 1 in parallel 8 columns (shorter / continuous feed) Wash 1 in series 6 columns (longer / discontinuous feed)

W1

Feed

2nd

Pass

W1

Feed 2nd

Pass Longer residence time in the elution zone Similar column cycling compared with protein A Productivity 3.7X batch process

SMB Transformation of Platform CEX Step

• 1.2cm x 3cm pre-packed columns • Poros HS Adsorbent • qbatch=50mg/mL • Feed = 11-13g/L • 2 different load zone configurations • Good agreement between experimental and

theoretical capture efficiency • CMCC loading was 60-73mg/mL at high yield

>95%

3.7 x Specific Productivity

⋅⋅−

−⋅

⋅=

SSSS1-1NTU-exp11111-1NTU-exp-1CE

Design Equations*

*Miyauchi and Vermeulen (1963)

++== ∑

1

21

WfeedfeedoL

i

ioL QQ

VNQ

VNakQ

VNakNTU0cQ

qQSfeed

BedBed

⋅⋅

=

Aggregate Clearance – Wash in Series Configuration

• Effect of column height investigated • 1.2 x 3.4cm, 1.2 x 6.8cm, 0.5 x 20cm • Feed aggregation varied (low and high) • Six 1.2 x 3.4cm columns for MCC • 4th cycle fractionation (20 fractions per column pooled)

• Similar pre-peak observed in batch and MCC Process

• Similar pool aggregate levels observed

• Little difference observed at different column heights

0%

2%

4%

6%

8%

10%

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

% A

ggre

gate

s

Normalized Elution Volume [-]

20cm High Agg20cm Low Agg6.8cm Low AggCMCC 3.4cm Low Agg

Integration of MCC CEX into Continuous DSP - 100L platform harvest

VI

BioSMB Protein A BRF

p p

Viral Filtration

Surge Bag

Surge Bag

Formulation: BRF/DiaF

Continuous UF

p

p

BioSMB CEX

A E X M

BRF

Surge Bag

S U B*

Depth /BRF

Filtration

Surge Bag

Surge Bag

p p

BRF p p

CRITICALITY

Continuous UF

-100%

-80%

-60%

-40%

-20%

0%

20%

40%

60%

80%

100%

0 20 40 60 80 100 120 140

Con

cent

ratio

n Fa

ctor

[-]

Membrane Loading [L/m2]

0 200 400 600 800 1000 1200 1400

pH

Time [Min]

VI Feed AEX Feed CEX Feed

pH

0

0.1

0.2

0.3

0.4

0.5

Abso

rban

ce [m

AU]

0

0.25

0.5

0.75

1

1.25

1.5

0 10

0.25

0.5

0.75

1

1.25

1.5

0 1 2 3 4 5 6

Abso

rban

ce [m

AU]

STDEV(%) Between Columns =1.01%

Continuous CEX Performance

• 16 Overlaid CEX Elution Profiles • AEXM Effluent Feed

Column

Average Yield

DNA* [ppm]

HCP [ppm]

Res. ProA [ppm] % Monomer

Centrifugation 97.3% N/S N/S N/S N/S DF/BRF 98.6% 30,515 383,300 N/S N/S

Protein A SMB 98.1% N/S N/S N/S N/S Viral Inactivation 100% 2 1,063 2.1 89.8% Anion Exchange

Membrane 98.8% <LOQ 82 1.5 99.0% Cation Exchange Chromatography 84.2% <LOQ 605 <LOQ 99.2%

SPTFF 99.5% 0.001 35 <LOQ 99.0% Overall 77.9% 0.001 8.7 <LOQ 99.0%

Continuous Processing Case Study mAb 2 - Platform

Mass Balance = 93%

DSP Productivity Enhancement

Step Continuous

Protein A Chromatography [g/(L·h)]

3.1

Cation Exchange Chromatography [g/(L·h)]

3.7

Overall [g/day]

~3x

• MCC steps enjoy a modest specific productivity increase

• Other steps suffer from lower specific productivities because they are slowed to accommodate the incoming flow rate

• The overall DSP will be 2-4x more productive (g/day) by operating in parallel (dependent on Protein A column sizing)

Conclusions and Future Work • A platform cation exchange step was transformed into a MCC

process – 3.6X specific productivity increase – Maintained consistent aggregate separation performance

compared to the batch process – Integrated into continuous DSP top reflect platform operation

with 84% yield at the 100L scale – Matched cycles with protein A step

• Interface CEX step with continuous viral filtration • Scale up process to 2000L in 24hours

Acknowledgements

• BTE – Ying Hou – David Pollard

• Analytical Support – Joe Fantuzzo – John Troisi – Jun Heo

• Fermentation Support – Patty Rose – Chris Kistler – Rachel Bareither

• Protein Purification Process Development – Nihal Tugcu – Thomas Linden

– Marc Bisschops – Steve Allen

Questions?