Merck Workshop - Platform Purification Strategies
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Transcript of Merck Workshop - Platform Purification Strategies
Platform purification strategies
Dr. Lothar Jacob
Page 2
Outline
• Monoclonal antibody production
• Protein A free antibody production
• Plasmid isolation
• Some examples of Vaccine purification
Page 3
Current Mab quantities
Although dosisis different,
Throughput is always an issue
3.2 mgIbritumomab tiuxetanZevalin®
25 mgDaclizumabZenapax®
50 mgPalivizumabSynagis®
20 mgBasiliximabSimulect®
100 mgRituximabRituxan®
10 mgAbciximabReoPro®
100 mgInfliximabRemicade®
5 mgMuromonab-CD3OKT-3®
5 mgGemtuzumab ozogamicinMylotarg®
440 mgTrastuzumabHerceptin®
30 mgAlemtuzumabCampath-1H®
DoseGeneric nameProduct
Page 4
Where is the bottleneck today?
• Drug-Target Search
• Upstream fermentation of mammalian cells?
• Downstream processing becomes a bottleneck
• Especially, the capture step is critical
capture20-100x
2-5x
Page 5
Generic protocols are not suitable
Because eachproduct
differs widely,platform technologies help
to solve the bottleneck
Generic dsp purification• One protocol fits every
Mab-candidate• No further optimization• Similar operational
conditions to different proteins
Individual dsp protocol• Various techniques• Different conditions• Different order of sequence
Page 6
One generic purification…..
• Amount of hcp
• Amount of DNA
• capacity & selectivity
• loading time
• number of cycles
• overall output• level of process
related impurities
• Aggregates & dimers
• Isoforms
• pI of Mab
• Content of Mab
• level of impurities
• yield
is not possible
Page 7
Platform Technology:
• Predetermined– Set of resins & buffers & salts / load capacity / loading flow rate
– Bed height (certain column diameters)
– Column regeneration & storage
– Type and volume of equilibration
– post load wash buffers
• Development needed for– Resin load capacity
– Wash II buffer (type & volume)
– Elution pH
– salt concentration
Page 8
Commercial ProductionPhase IIIPhase IIPhase IPre-clinical
Clone1, repeated batchExtended batch
6 x optimized batchFormulation 2Pre-filling syringes
Process enhancementsoptimized batchFormulation 2
Clone2Repeated batchFormulation 1
1,000Loperational
6,000Lfacilitydecision
3,000Loperational
2nd 3,000Loperational
RegistrationRuns,12,000LDecision
EU 3KApproval US6K approval
12,000L Mech.complete
2005200420032002200120001999199819971996
Humira: Development timeline
US 3K approvalSource: Downstream – Gab ‚04 abstracts
Page 9
Comparison of different production processes
HerceptinTM Rituxan MabCampathTM SynagisTM RemicadeTM SimulectTM
Cell removal 1 1 1 1 1 1Protein A Affinity chromatography 2 2 2 2 2
Virus inactivation 3 3 3 4 3 3
Cation exchange 4 5 4 3 4 5Anion exchange 5 4 2 & 6 6 & 7 4Hydrophobic interaction 6
Size exclusion chromatography 5 8
Virus clearance 6 6 5 & 6 5 6Sterile filtration 7 7 7 9 8 7
The numbers indicate the position of the step within the dsp processing scheme.
modified acc. to Sommerfeld & Strube, Chemical Engineering and Prosessing 44, 1123-1137, 2005
Page 10
Cell free supernatant
Pure antibody
BI’s current production strategy
Prot A
CEX
AEX HICAEX HIC
Flow Through mode
capture
• Scale: 5000-20,000L
• Titers 0.5 – 6 g/L
• Batches of 60-100 kg
• Prot. A binding capacity~ 35 g/L;2 m ∅ column, bed height 15 cm
• IEX binding capacity~ 40-100 g/L,1.5 m ∅, bed height 25 cm
intermediate
polishing
Page 11
Merck Serono Biotech Center
Platform fits to equipment?
• Does the process fit to the purification suite?
• Has the manufacturing site to be engineered newly
Page 12
Training classes on column packing
Flow rate during packing 8.5 L/min, 347 cm/hr, conditioned at 20 psi
Page 13
Platform published by Amgen
Cell culture harvest
Pure antibodybatches >10kg
Prot A chromatography
Cation exchanger
•Equilibration >5•Direct load of ccs•Intermediate pH wash•Low pH elution
hcp removal, leached Prot. A,Aggregate removal Anion exchanger/
Hydroxyapatite/HIC
Cell culture harvest
Pure antibodybatches >10kg
UF/DF
Prot A
Polishing
Processsafety;robustness
2x10² hcpreduction
•Glycine Glycinate/NaCl, pH 8.0•DBC=20-35g/L
Page 14
Platform published by BiogenIdec
Clarified conditioned media
Phenyl HIC-Step
rProt A
Anion exchanger (binding mode)
Low pH virus inactivation
Planova 15 VF
88% purity
UF/DF
Reduction of incomplete Ab to <2%Reduction of aggregates to <2.7%Removal of high pI isoforms
Reduction of aggregates to <1%Reduction of incomplete Ab to <1%
Wolf Noe, BioProcess Development Biogen-IdecBioProduction IBC meeting, Paris 2007
Page 15
Platform strategy of Wyeth
Cell culture harvest
UF/DF
Prot A
Anion exchanger (binding mode)
# of re-cyclingcolumn size
throughputprocess economy
Virus filter
Limitations of Protein A:• High resin cost
• Residual Protein A impurity
• Sanitisation under harshconditions results in loss of capacity
• Acid elution may result in aggregation for someantibodies
Source: Poster WilBio 2006, Thousand Oaks, Packed Bed Hydrodynamics of a HighlyCharged Ion Exchange Resin at Ionic Strength Extremes; Aaron Noyes et al.
Page 16
Cell free supernatant
Pure antibody
Cation exchanger
Prot A
Anion exchanger
Medimmune’s Improved strategies
UF/DF
Nanofiltration/Low pH inactivation
Benzonase®
Sterile filtration
Shane & Oliver, PDA Conference (Berlin, september 2001)
Page 17
TFF
CHT (20 mg/ml)
CEX (15-20 mg/ml)
viral inactivation
AEX (15mg/ml);FT mode
Virus filter
TFF
CEX (40-100mg/ml)
viral inactivation
Virus filter
AEX(50mg/ml)FT mode
Q-Membrane(2100 mg/ml)
FT mode
recovery: 85-95%
Formulation TFF
mab level ~4g/L
Medarex‘ Platform without Protein A
Platform
Suppl. to Biopharm International, February 2007
Page 18
Medarex Technology Platform
Old PlatformUpstream
Hybridoma
Perfusion
40-60 mg/L
Harvest by TFF
DownstreamOnly Protein A processes
3 chromatography steps
Multiple in-process TFFs
IV formulations
Alahari Arunakumari, Paris, 24th & 25th April 2007
Current Platform Upstream
CHO
Fed-batch
> 3 g/L
Harvest by DepthF
DownstreamMainly Non-ProteinA
2 chromatography steps
No in-process TFFs
IV, ID, and subQ formulations
BioProcess International European Conference and Exhibition,
Page 19
Upgrade Potential productivity
Protein Asoftgel VI UF IEX UFVRF IEX UF
VI UF UFVRF UFProtein Arigid IEX time reduction
-44%
IEX
for increased productivity rigid media are advantageousHigher bed height = more column capacity = less re-cycling
From: An evaluation of Protein A and Non-Protein A methods for the recovery of monoclonal antibodies and considerations for process scale-up by Martin Smith, LONZA, Presented at“Scaling-up of Biopharmaceutical Products”, 26/27thJanuary 2004, The Grand, Amsterdam
Page 20
High throughput screening for binding capacities on ion-exchange
Part I: batch capacity experiments for CEXcapacity (mg/ml)
25
35
Fractoge
l®EMD SE, p
H6
Toyope
arl® SP, p
H6
Frac
togel
® EMD SO 3
- , pH
6
SP Sepharo
se® FF
, pH
5
SP Sepharo
se® FF
, pH
6
SP Sepharo
se® XL,
pH5
SP Sepharo
se® XL,
pH6
CM Sepharo
se® FF
, pH
6
Toyope
arl® CM, p
H6
Macro
prepHS, p
H6
15
5
All data from: Generic purification processes for monoclonal antibodies and Fc fusion proteins; Abhinav Shukla, Peter Hinckley, Eva Gefroh, Priyanka Gupta and Brian Hubbard, Amgen, IBC meeting 2002, San Diego, USA
Page 21
Higher Resolution for Polishing
0
500
1000
1500
2000
2500
3000
mAU
4.2
4.4
4.6
4.8
5.0
5.2
5.4
5.6
20.0
40.0
60.0
80.0
mS/cm
0 50 100 150 minX1 X2 X31A11A111B41C5 1C6-1F6 1F51G4X4 Waste 0
500
1000
1500
2000
2500
3000
mAU
4.5
5.0
5.5
20.0
40.0
60.0
80.0
mS/cm
0 50 100 minX1 X2 X31A11A111B41C5 1E31E121F41G5X4 Waste
Fractogel® SO3 (M)Particle size 40-90µm
Fractogel® SO3 (S)Particle size 20-40µm
pH 5
Comparison of S- and M-Type Resins
Page 22
Example chromatograms (pH 6)
CEX 1 CEX 2Sharp elution is preferred
mAb pool
0
500
1000
1500
2000
2500
3000
3500
mAU
0 50 100 150 200 250 ml
mAb pool
0
1000
2000
3000
mAU
0 50 100 150 200 250 ml
Page 23
Development of a purification processwithout Protein A
CEX capture step:
• Today’s cell culture harvests are cleaner (fewer protein impurities) than in the past when serum and/or proteins were common additives: reduced requirement for affinity purification
• Most antibodies have a higher isoelectric point than their protein contaminants: direct capture by CEX should provide HCP clearance
• CEX resins are reasonably priced, they can be sanitised under harsh conditions, some provide high capacities at high flow rates
Page 24
Screening methods: batch tests
24
10-200 µl resin suspension
Automated preparation of 96-deep-well resin plate=>small amount of gel and sample allows a large number of tests
Page 25
Parameters to be investigated
Parameter plate columnBinding capacity static dynamic Yield (yes) yes
Biological activity yes yes
Purity hcp
Prot. A leakage
aggregates
Washing conditions (yes) yes
cycle number no yes
Page 26
Pros and Cons: Multiwell plates
• Advantage: Many conditions can be tested
• Disadvantage: Additional characterization of many samples is laborious
• Static vs dynamic binding capacity
• Column effects
• Scale-dependent parameters
Page 27
Process steps of pDNA purification
Improved downstream process for the production of plasmid DNA for gene therapy, Jochen Urthaler, Wolfgang Buchinger and Roman Necina; Acta Biochimica Polonica Vol. 52 No. 3/2005, 703–711
Anion Exchange Chromatographyon Fractogel EMD DEAE, dynamic binding capacity between5 and 10 g pDNA/l
HIC step can be directly usedas feed solution for the AECHIC
AEX
SEC
Fermentation
Alkaline Lysis
Conditioning*
*Adjustment forbinding on HIC (AS) Adjustment of conc.
(UF/dilution
Page 28
Adenovirus production process
Amine Kamen and Olivier HenryDevelopment and optimization of an adenovirus production process, J Gene Med 6, S184–S192, 2004
Adenovirus production
HarvestLiquid
Cell lysis
Benzonase treatment/Centrifugation
Anion Exchange Chromatography on Fractogel EMD DEAE
Solid
Filtration
Ultrafiltration/Concentration
Ret
enta
te(a
deno
viru
s) Size ExclusionChromatography
PurifiedAdenovirus
Page 29
Purification of rec. Adenovirus onFractogel® EMD DEAESample preparation:Viruses was propagated in 293 cells, release is achieved by three cycles of freeze/thaw of washed cells after centrifugation or directly; Benzonase(final conc. 100 U/ml) was added for 30 min at r. t.
Chromatography:Viral lysate was loaded onto a 3x6 cm Fractogel® EMD DEAE (M)column equilibrated with 50 mMTRIS/HCl, 100 mM NaCl, 2 mMMgCl2, 2% sucrose; pH 8Sequential washes were performedat 0.1 and 0.2 M NaCl
Elution:Bound virus was then elutedat 0.35 M NaCl
Data from: Puresyn, Inc.; Malvern, PA
virus
time
A260
0.1 M
0.25
M 0.
35 M
1 M
0.5
M N
aOH
Page 30
Purification strategy
Lysate Benzonase treatment
Cell lysis
Fractogel® EMD DEAE
SEC Polishing
Purified Ad5
Centrifugation/Lysate supernatant conditioning
Concentration
Filter
75-95%
60-70%
45-65%
15-30%
85-100%
20-35%
Average Overall
Recovery
process
Adenovirus Type 5 (Ad5) chromatographicpurification process at the 20 L scale; Arcandet al., BioProcessing Journal, Jan/Feb. 2003
Page 31
Purification of Alphavaccines
Virus-like replicon particle (VRP) capture by AEX based operation
Electroporation
filtration on Sartopore 2 (0.45/2µ)
Formulation
TFF purification and Benzonase treatment
Cellufine Sulfate (replaces Heparin
Sepharose FF)
process
Development and manufacture of alphavaccines; T. Talarico et al., BioProcessing Journal, Fall 2006
Example: VAQTA® production
No nucleasetreatment
Nuclease treatmentafter capture step
Nuclease treatmentafter harvest
LysateCapture
chromatography
PEG-precipitation
LsyateCapture
chromatographyNucleasetreatment
PEG precipitation
LysateNucleasetreatmentCapture
chromatographyPEG precipitation
HAV recovery 30 % 36 % 57 %
ratio:HAV/protein
1.7 1.8 5.0
ratio:HAV/DNA
1.000 >42.500 >42.500
Page 33
~8mL “Bioreactor”
~20L of “Bioreactors
Simon Hsu: Case Study: Establishing a Benchmark for Economic Vaccine Scale-up StrategiesMedImmune Vaccines, Inc., European BioPharm Scale-Up Congress 2008, 17-19 September 2008, Geneva, Switzerland
Current Egg-based manufacturing
Page 34
• Benzonase® treatmentInefficient DNA degradation in the presence of virus;
high MW DNA remaining
• Benzonase® treatment followed by tangential flow filtrationInefficient removal of high MW DNA by TFF
• Benzonase® treatment followed by column chromatographyHigh MW DNA removed
Simon Hsu: Case Study: Establishing a Benchmark for Economic Vaccine Scale-up StrategiesMedImmune Vaccines, Inc., European BioPharm Scale-Up Congress 2008, 17-19 September 2008, Geneva, Switzerland
Approaches used to remove DNA
Page 35
Viral particles (VP): measured by HPLC analytical anion-exchange assay; infectious units (IU): measured by tissue culture infectious dose (TCID50) assay; purity: determined by SDS-PAGE and Western blot analysis or by integration of HPLC chromatogram at 260 nm.
(modified according to Burova & Ioffe, Gene Therapy 12, 2005
culture productionSephacryl S-400 HR2. Size exclusion
Kamen and Henry (2004)
99Final product
8020 l scale suspensionFractogel EMD DEAE (M)1. Anion exchange
5447Final product
9778PolyFlo2. Proprietary resin
90731015 input viral particlesFractogel DEAE (M)1. Anion exchange
5755Final product
9484PolyFlo2. Proprietary resin
75731014 input viral particlesFractogel DEAE (M)1. Anion exchangeGreen et at (2002)
254099Final product
Source 15Q2. Anion exchange
811013 input viral particlesQ Sepharose XL1.Anion exchangeBlance et al (2000)
2232Final product
88444798IMAC column with zinc/glycine system2. Affinity
824967921013 input viral particlesFractogel DEAE-650 M1. Anion exchangeHuyghe et al (1995)
VP/IUratio
Yield(IU, %)
Yield(VP, %)
Purity(%)
Purification scaleColumn typeChromatographicpurification steps
Study
Efficiencies of Ad chromatographic steps
Page 36
Future trends…?
H. Graalfs Page 13
1
10
100
1000
Prot A IEX Max.Cap.
pH
Bin
ding
cap
acity
Chromatography mode
Screening! Automation
Salt
Solubility of MAbPrecipitation during elutionViscosityAggregation
Protein titer: 0.2 – 10 mg/mL
mAb crystallization !?
IgG
IgG
BSA
Page 37
Page 38
Thank you