Platform purification strategies

Dr. Lothar Jacob

Outline

Monoclonal antibody production Protein A free antibody production Plasmid isolation Some examples of Vaccine purification

Page 2

Current Mab quantities

Product
Campath-1H Herceptin

Generic name
Alemtuzumab Trastuzumab Gemtuzumab ozogamicin Muromonab-CD3 Infliximab Abciximab Rituximab Basiliximab Palivizumab Daclizumab Ibritumomab tiuxetan

Dose
30 mg 440 mg 5 mg 5 mg 100 mg 10 mg 100 mg 20 mg 50 mg 25 mg 3.2 mg
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Although dosis is different, Throughput is always an issue

Mylotarg OKT-3 Remicade ReoPro Rituxan Simulect Synagis Zenapax Zevalin

Where is the bottleneck today?

Drug-Target Search

Upstream fermentation of mammalian cells?

1 02

0x 0

capture

Downstream processing becomes a bottleneck 2-5x

Especially, the capture step is critical

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Generic protocols are not suitable

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

Because each product differs widely, platform technologies help to solve the bottleneck
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One generic purification..

pI of Mab Content of Mab Amount of hcp Amount of DNA Aggregates & dimers Isoforms capacity & selectivity loading time number of cycles overall output level of process related impurities level of impurities yield

is not possible
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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
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Humira: Development timeline

Clone1, repeated batch Extended batch Clone2 Repeated batch Formulation 1 optimized batch Formulation 2 6 x optimized batch Formulation 2 Pre-filling syringes Process enhancements

Pre-clinical

Phase I Phase II

Phase III

Commercial Production 2002 2003 2004 2005

1996

1997

1998

1999

2000

2001

1,000L 3,000L 6,000L operational operational facility decision

Source: Downstream Gab 04 abstracts

2nd 3,000L operational

Registration Runs, 12,000L Decision

EU 3K Approval US 6K approval 12,000L Mech. complete

US 3K approval

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Comparison of different production processes

HerceptinTM Cell removal Protein A Affinity chromatography Virus inactivation Cation exchange Anion exchange Hydrophobic interaction Size exclusion chromatography Virus clearance Sterile filtration 1 2 3 4 5 6

Rituxan 1 2 3 5 4

MabCampathTM 1 2 3 4

SynagisTM 1

RemicadeTM 1 2

SimulectTM 1 2 3 5 4

4 3 2&6

3 4 6&7

5 7 6 7 6 7

8 5&6 9 5 8 6 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
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BIs current production strategy

Scale: 5000-20,000L Titers 0.5 6 g/L Batches of 60-100 kg

Cell free supernatant

Prot A

capture

Prot. A binding capacity ~ 35 g/L; 2 m column, bed height 15 cm IEX binding capacity AEX ~ 40-100 g/L, AEX 1.5 m , bed height 25 cm

CEX

intermediate

HIC HIC

polishing

Pure antibody

Flow Through mode

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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 11

Training classes on column packing

Flow rate during packing 8.5 L/min, 347 cm/hr, conditioned at 20 psi
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Platform published by Amgen

Cell culture harvest Cell culture harvest
Glycine Glycinate/NaCl, pH 8.0 DBC=20-35g/L

UF/DF

Prot A Process safety; robustness

Prot A chromatography 2x10 hcp reduction Cation exchanger

Equilibration >5 Direct load of ccs Intermediate pH wash Low pH elution

Polishing

Anion exchanger/ Hydroxyapatite/ HIC

hcp removal, leached Prot. A, Aggregate removal

Pure antibody batches >10kg

Pure antibody batches >10kg

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Platform published by BiogenIdec

Clarified conditioned media

rProt A
Anion exchanger (binding mode)

88% purity Reduction of incomplete Ab to <2% Reduction of aggregates to <2.7% Removal of high pI isoforms

Low pH virus inactivation

Phenyl HIC-Step Planova 15 VF UF/DF

Reduction of aggregates to <1% Reduction of incomplete Ab to <1%

Wolf Noe, BioProcess Development Biogen-Idec BioProduction IBC meeting, Paris 2007
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Platform strategy of Wyeth

Cell culture harvest

Limitations of Protein A:
High resin cost Residual Protein A impurity Sanitisation under harsh conditions results in loss of capacity Acid elution may result in aggregation for some antibodies

Prot A

# of re-cycling column size

Anion exchanger (binding mode)

throughput process economy

Virus filter UF/DF

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Source: Poster WilBio 2006, Thousand Oaks, Packed Bed Hydrodynamics of a Highly Charged Ion Exchange Resin at Ionic Strength Extremes; Aaron Noyes et al.

Medimmunes Improved strategies

Cell free supernatant

Cation exchanger
Benzonase

Prot A
Nanofiltration/Low pH inactivation

Anion exchanger

UF/DF
Sterile filtration

Pure antibody

Shane & Oliver, PDA Conference (Berlin, september 2001)

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Medarex Platform without Protein A

TFF CEX (15-20 mg/ml)
mab level ~4g/L

TFF

Platform

CEX (40-100mg/ml)

recovery: 85-95%
viral inactivation viral inactivation

AEX (15mg/ml); FT mode

CHT (20 mg/ml)
Virus filter
Formulation TFF

AEX (50mg/ml) FT mode

Q-Membrane (2100 mg/ml) FT mode

Virus filter
Suppl. to Biopharm International, February 2007
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Medarex Technology Platform

Old Platform
Upstream Hybridoma Perfusion 40-60 mg/L Harvest by TFF

Current Platform
Upstream CHO Fed-batch > 3 g/L Harvest by DepthF

Downstream
Only Protein A processes 3 chromatography steps Multiple in-process TFFs IV formulations
Alahari Arunakumari, Paris, 24th & 25th April 2007

Downstream
Mainly Non-ProteinA 2 chromatography steps No in-process TFFs IV, ID, and subQ formulations
BioProcess International European Conference and Exhibition, Page 18

Upgrade Potential productivity

Protein A softgel

VI UF IEX UF VRF

IEX

UF

Protein A rigid

VI UF

UF VRF IEX UF

time reduction -44%

for increased productivity rigid media are advantageous Higher 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 atScaling-up of Biopharmaceutical Products, 26/27thJanuary 2004, The Grand, Amsterdam
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IEX

High throughput screening for binding capacities on ion-exchange

Part I: batch capacity experiments for CEX capacity (mg/ml) 35 25 15 5
To FF yo ,p pe H 6 ar l CM To ,p yo H 6 pe ar Fr l ac SP to ,p ge H l 6 EM Fr D ac SE to ,p ge H l 6 EM D SO M ac 3 , ro pH pr 6 ep HS ,p H 6 6 5 5 6 CM Se ph a XL ,p H
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

pH FF ,

ro se

XL ,p H

FF ,

pH

ro se

ro se

Se ph a

Se ph a

SP

SP

Se ph a

SP

SP

Se ph a

ro se

ro se

Page 20

Higher Resolution for Polishing

mAU mS/cm 5.6 3000 80.0

mAU mS/cm 3000 80.0 5.5 5.4

Fractogel

SO3 (S)

2500 5.2 60.0 2000 5.0 1500 40.0 4.8

2500

Fractogel SO3 (M)

60.0

Particle size 20-40m

2000

Particle size 40-90m

5.0

1500 40.0

1000 4.6 20.0 500 4.4

1000

20.0 500

4.5

0 0

X1

X2 50

X31A1 1A11 1B41C5 1C6-1F6 1F5 1G4X4 Waste4.2 100 150 min

0 0

X1

X2

X31A11A11 1C5 1B4 1E3 1E12 1G5X4 Waste 1F4 50 100 min

pH 5

Comparison of S- and M-Type Resins

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Example chromatograms (pH 6)

mAU mAU

3500

3000 3000

2500

2000

2000

1500

1000

1000

500

mAb pool
0 0 50 100 150 200 250 ml 0 0 50 100

mAb pool

150

200

250

ml

CEX 1

CEX 2
Sharp elution is preferred
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Development of a purification process without Protein A

CEX capture step:
Todays 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

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Screening methods: batch tests

Automated preparation of 96-deep-well resin plate => small amount of gel and sample allows a large number of tests

10-200 l resin suspension

24
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Parameters to be investigated
Parameter
Binding capacity Yield Biological activity Purity

plate
static (yes) yes hcp

column
dynamic yes yes

Prot. A leakage aggregates Washing conditions cycle number (yes) no yes yes
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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

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Process steps of pDNA purification

Fermentation HIC Alkaline Lysis AEX

HIC step can be directly used as feed solution for the AEC Anion Exchange Chromatography on Fractogel EMD DEAE, dynamic binding capacity between 5 and 10 g pDNA/l

SEC Conditioning*
*Adjustment for binding on HIC (AS)

Adjustment of conc. (UF/dilution

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, 703711
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Adenovirus production process

Harvest Adenovirus production Benzonase treatment/ Centrifugation Liquid Cell lysis Solid Filtration Retentate (adenovirus) Size Exclusion Chromatography

Anion Exchange Chromatography on Fractogel EMD DEAE

Purified Adenovirus

Ultrafiltration/ Concentration

Amine Kamen and Olivier Henry Development and optimization of an adenovirus production process, J Gene Med 6, S184S192, 2004

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Purification of rec. Adenovirus on Fractogel EMD DEAE

Sample 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 mM TRIS/HCl, 100 mM NaCl, 2 mM MgCl2, 2% sucrose; pH 8 Sequential washes were performed at 0.1 and 0.2 M NaCl Elution: Bound virus was then eluted at 0.35 M NaCl
Data from: Puresyn, Inc.; Malvern, PA A260

0.1 M

0.25 M

0.35 M

1M

virus

time

0.5 M NaOH
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Purification strategy
process Cell lysis 60-70% Average Overall Recovery

Lysate Benzonase treatment

75-95%

Centrifugation/Lysate supernatant conditioning

85-100%

Filter
Fractogel EMD DEAE 45-65%

Concentration

20-35%

SEC Polishing

15-30%
Adenovirus Type 5 (Ad5) chromatographic purification process at the 20 L scale; Arcand et al., BioProcessing Journal, Jan/Feb. 2003

Purified Ad5

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Purification of Alphavaccines
process Electroporation filtration on Sartopore 2 (0.45/2) Virus-like replicon particle (VRP) capture by AEX based operation

TFF purification and Benzonase treatment

Cellufine Sulfate (replaces Heparin Sepharose FF)

Formulation

Development and manufacture of alphavaccines; T. Talarico et al., BioProcessing Journal, Fall 2006

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Example: VAQTA production

No nuclease treatment Lysate Capture chromatography

Nuclease treatment Nuclease treatment after capture step after harvest Lsyate Capture chromatography Nuclease treatment PEG precipitation 36 % 1.8 >42.500 Lysate Nuclease treatment Capture chromatography PEG precipitation 57 % 5.0 >42.500

PEG-precipitation HAV recovery ratio: HAV/protein ratio: HAV/DNA 30 % 1.7 1.000

Current Egg-based manufacturing

~8mL Bioreactor

~20L of Bioreactors

Simon Hsu: Case Study: Establishing a Benchmark for Economic Vaccine Scale-up Strategies MedImmune Vaccines, Inc., European BioPharm Scale-Up Congress 2008, 17-19 September 2008, Geneva, Switzerland Page 33

Approaches used to remove DNA

Benzonase treatment Inefficient DNA degradation in the presence of virus; high MW DNA remaining Benzonase treatment followed by tangential flow filtration Inefficient removal of high MW DNA by TFF Benzonase treatment followed by column chromatography High MW DNA removed
Simon Hsu: Case Study: Establishing a Benchmark for Economic Vaccine Scale-up Strategies MedImmune Vaccines, Inc., European BioPharm Scale-Up Congress 2008, 17-19 September 2008, Geneva, Switzerland
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Efficiencies of Ad chromatographic steps

Study Huyghe et al (1995)

Chromatographic purification steps 1. Anion exchange 2. Affinity Final product

Column type Fractogel DEAE-650 M IMAC column with zinc/glycine system

Purification scale 1013 input viral particles

Purity (%)

Yield (VP, %)

Yield (IU, %)

VP/IU ratio

92 98

67 47 32

49 44 22

82 88

Blance et al (2000)

1.Anion exchange 2. Anion exchange Final product

Q Sepharose XL Source 15Q

1013

input viral particles

81

99
Fractogel DEAE (M) PolyFlo 1014 input viral particles

40 73 84 55 75 94 57 90 97 54

25

Green et at (2002)

1. Anion exchange 2. Proprietary resin Final product 1. Anion exchange 2. Proprietary resin Final product

Fractogel DEAE (M) PolyFlo

1015 input viral particles

73 78 47

Kamen and Henry (2004)

1. Anion exchange 2. Size exclusion Final product

Fractogel EMD DEAE (M) Sephacryl S-400 HR

20 l scale suspension culture production

80

99

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
Page 35

Future trends?
Binding capacity 1000

mAb crystallization !? Screening! Automation

BSA

100

IgG

pH
IgG

10

Solubility of MAb Precipitation during elution Viscosity Aggregation

Salt
Protein titer: 0.2 10 mg/mL

1 Prot A
H. Graalfs

IEX

Max. Cap.

Chromatography mode

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Thank you

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