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