platform downstream processes in the age of continuous chromatography: a case study
DESCRIPTION
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. Transition to Future Concepts. - PowerPoint PPT PresentationTRANSCRIPT
Platform downstream processes in the age of continuous chromatography: A case study
Mark Brower
BioProcess Technology & ExpressionBioprocess Development
Kenilworth, NJ
Integrated Continuous BiomanufacturingCastelldefels, Spain20-24 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 PurificationProtein A Chromatography
Viral Inactivation(Low pH Hold)
DNA / HCP / Viral AdsorptionAnion Exchange Chromatography
Variant and Aggregate ClearanceCation Exchange Chromatography
Viral FiltrationNanofiltration
Concentration / Buffer ExchangeMicrofiltration / Diafiltration
Bioburden ReductionSterile Filtration
mAb Downstream PurificationBulk
Formulation
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 StepOther Steps are Lengthened to Compensate
S U B*
Depth /BRF
Filtration
SurgeBag
BioSMB Protein A
Single-UseCentrifugation
SurgeBag
p p
BRF
pp
BRF
pp
Viral Filtration
SurgeBag
SurgeBag
Formulation: BRF/DiaF
Continuous UFAnion
ExchangeMembrane
p
p
Polishing Step
Continuous Viral Inactivation
AEXM
BRF
SurgeBag
*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
HarvestBag
DF / BRF
SU CentrifugeSMB Protein A
Viral Inactivation
AEX Membrane
Mixed Mode
SPTFF
Continuous Processing Case Study mAb 1- Non-platform
MCC for Bind & Elute Applications
Eq
Waste
2 nd pass
Depleted Feed
2nd pass
Was
h
Elu
te Produ
ct
Feed
Strip
Was
h
2
C1
C5
C2
C6
C3
C7
C4
C8
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
PassLonger 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-exp1
11
11
-1NTU-exp-1CE
Design Equations*
*Miyauchi and Vermeulen (1963)
1
21
WfeedfeedoL
i
ioL QQ
VN
Q
VNak
Q
VNakNTU
0cQ
qQS
feed
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
Integration of MCC CEX into Continuous DSP- 100L platform harvest
VI
BioSMB Protein A
BRF
pp
Viral Filtration
SurgeBag
SurgeBag
Formulation: BRF/DiaF
Continuous UF
p
p
BioSMB CEX
AEXM
BRF
SurgeBag
S U B*
Depth /BRF
Filtration
SurgeBag
SurgeBag
p p
BRF
pp
CRITICALITY
Continuous UFpH
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/SDF/BRF 98.6% 30,515 383,300 N/S N/S
Protein A SMB 98.1% N/S N/S N/S N/SViral 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?