D o w n s t r e a m 2 7 In this issue · In timely fashion, FPLC, the system which ... For fast information about the new standard in protein purification, cut out the form ... Malaysia603

Custom-designed solutions expand engineering operationsNew engineering facilities have been built to help meet the growing interest in tailor-made solutions for large-scale columns and systems. The new facilities are purpose-designed and include a new product management system which will facilitate the pathfrom specification to final product.

ÄKTAFPLC-the new standard in protein purificationInterest in proteins has never been greater as research swings from describing thegenome to studying its expressed products. In timely fashion, FPLC, the system whichrevolutionised protein purification in the 80’s, has received its biggest ever update.ÄKTAFPLC combines all the advantages of the ÄKTAdesign platform with theestablished biocompatibility and reliability of FPLC, making it well prepared to meet thechallenges of modern research.

STREAMLINE 100 – mid-range expansionAn increase in the range of column dimensions means that there is now aSTREAMLINE column for capturing product from bioreactors with volumesup to 100 litres.

Towards large-scale purification of plasmid DNA and a viral vector intended forhuman gene therapy Delivering genetic material safely and effectively to its target cell is a major challenge.The use of plasmids and modified viruses are just two potential transport systems underinvestigation. If these are to be viable delivery vehicles, then they must be able to bepurified in large quantities. This article reports on two scalable purification methods.

Methods of inactivation of transmissible spongiform encephalopathies (TSEs) asapplied to production chromatography processesMad-cow disease increased public awareness of infections derived from TSEs. Thecausative agent is believed to be the prion protein. Removing this tenacious and resilientprotein has proved difficult. This article reviews the most successful methods used todiminish its infectivity.

Meeting reports– Conference on Affinity Chromatography: April 16-17, 1998,San Diego, California, USA.

– International conference on vectors for gene therapy: design, production andregulation: March 12-14, 1998, Brussels, Belgium.

ÄKTAFPLC faxbackFor fast information about the new standard in protein purification, cut out the formand fax to your nearest office.

Fast Trak Course detailsCourse details from the Fast Trak Centers around the world.

Head office Sweden 46 (0)18 16 50 00 • Australia 1 800 222 216 • Austria 01 68 66 250 • Belgium 3 272 14 69 • Brazil 55 11 872 68 33 • Canada 1 800 463 5800 • Denmark 48 14 10 00 • Eastern Europe 43 1 982 38 26 • Far East 852 2811 86 93 • Finland 358 9 512 39 40 • France 0 1 69 35 67 00 • Germany 07 61 49 030 • Great Britain 01 727 81 40 00 • The Netherlands 0165 580 400 • Hong Kong 852 2811 8693 • India 91 44 434 0747

• Italy 02 273 221 • Japan 03 5992 3426 • Malaysia 603 724 20 80 • Middle East 30 1 960 0687 • New Zealand 0 800 733 893 • Norway 63 89 23 10 • Peoples Rep. of China 852 2811 8693 • Portugal 01 417 7035• Republic of Korea 82 2 511 0801 • Russia & CIS 7 (503) 956 1137 • Spain 93 589 07 01 • Sweden 018 164 000 • Switzerland 01 802 81 50 • Taiwan 886 2 831 5310 • United States 1 800 526 3593

(9806)

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D o w n s t r e a m 2 7

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In this issue

Front coverThe front cover shows our new engineeringfacilities in Uppsala, Sweden.

TrademarksÄKTA, BioProcess, CHROMAFLOW,Downstream, Fast Trak, FineLINE, FPLC,HiLoad, HiPrep, HiTrap, MONO Q, PhastGel,PhastSystem, RESOURCE, Sepharose,SOURCE, STREAMLINE, Superloop, Superdex,UNICORN, are trademarks owned byAmersham Pharmacia Biotech Limited or itssubsidiaries..

Addresses

Amersham Pharmacia Biotech UK Limited

Amersham Place

Little Chalfont

Buckinghamshire

England HP7 9NA

Amersham Pharmacia Biotech AB

SE-751 84 Uppsala

Sweden

Amersham Pharmacia Biotech Inc.

800 Centennial Avenue

PO Box 1327

Piscataway

NJ 08855 USA

Terms and Conditions of Sale

All goods and services are subject to the terms and conditions of sale of thecompany within the Amersham Pharmacia Biotech group which supplies them. Acopy of these terms and conditions is available on request.

© Amersham Pharmacia Biotech AB 1998– All rights reserved

27

Custom-designedsolutionsexpandengineering

operations

Increasing demands for custom-designed solutions, together with thedevelopment of new product lines, has lead to the engineering department in

Uppsala requiring larger premises. The department, which handles large-scalecolumn and system orders, recently acquired new design and testing facilities

to enable them to meet customers’ growing needs.

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Interest in new and innovativeproducts such as CHROMAFLOWcolumns, and STREAMLINE systems

and columns for expanded bedadsorption, is growing rapidly. For theengineering department in Uppsala, thisgrowth has meant a move into larger andmore modern facilities for testing anddeveloping product lines. New facilitieswere also a solution to meeting theincreasing trend for tailor-made solutions.

One of the factors contributing to thegrowing interest in large-scale columnsand systems is the stage many customershave reached with drug development.Many pharmaceutical companies are nowproceeding from clinical trials at pilot-scale towards production and thus requiremanufacturing-scale equipment. Thisincreasingly involves custom-designedsolutions.

Close to customerDeveloping tailor-made products involvesclose co-operation with the customer. Andwhat customers ask for can varytremendously: one customer may simplysay that he needs to purify a certainsubstance and ask for a suitable solution.Another may send a thick loose-leafbinder with complete specifications downto a very detailed level where every nutand bolt is carefully defined. This putsvastly different demands on the team in

Uppsala. In the first instance, it isnecessary to understand the customer’sapplications requirements and translatethem into suitable hardware. In thesecond instance, it requires in-depthknowledge of mechanics and materialssciences in order to interpret thecustomer’s specifications and, wherenecessary, to suggest alternatives.

Established proceduresSo how does the engineering departmentmeet these differing demands? Thedepartment has acquired a great deal ofexperience over the years, havingdelivered more than 700 systems and5000 columns world-wide. Based on this

experience, it has established routines fordeveloping a custom-designed system orcolumn. The path to final product closelyinvolves the customer, and includesextensive quality control and functionaltesting along the way, in addition to thatdone during installation. From placementof an order to delivery takesapproximately 4–7 months per system.This time being very much dependent onthe complexity of the customer’sspecifications.

Multi-discipline expertise Initial contact with a customer usuallycomes via a sales specialist, whereupon aproject manager is appointed in Uppsala.

Custom-designedsolutionsexpandengineering

operations

Choice of materialsMaterials traditionally used inpharmaceutical production include highquality stainless steel of grade 316L orequivalent. However, there are severalalternatives to stainless steel which offerexcellent resistance to salts and otheragents used in biopharmaceuticalproduction; these include polypropylene,Hastelloy* and PVDF. Plastic materialslike polypropylene are robust, chemicallyresistant and often more cost-effective.Today about 60% of systems aremanufactured in polypropylene.

Specialist assemblyAssembly of system and columncomponents usually takes place at one ofour qualified subcontractors. Throughoutthe assembly period workshop inspectionsare frequently carried out by the projectmanager to assure quality ofworkmanship.

When the mechanical and electricalassembly of a system has been completed,the system is brought to Uppsala forsoftware configuration, comprehensivefunctional testing, final adjustments andcalibration. Customers are also invited tocome to Uppsala to perform their own

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The project manager, together withengineers and specialists in Uppsala andour regional offices, carefully studies thecustomer’s specifications. This procedurecan be extremely demanding, requiringexpertise from many disciplines. Typicalareas for clarification are where anapplication requirement must match withchoice of materials and economy. Theproject manager will note all deviationsfrom the customer’s request and wherepossible suggest any alternatives. Thisphase, when completed, results in acomprehensive documentation of therequested individual specificationstogether with all comments, suggestionsand alternatives. With the finalspecifications determined, the customerformally places an order and design plansare drawn.

testing or inspection before delivery of thesystem. The system is again thoroughlytested at the customer’s site to assure it isperforming as specified. Thedocumentation binder, which containsevery piece of documentation accrued tothe system, is handed over to the customerafter completed installation and testing.

The flow path describes the stages from concept to

installation and operation of a system. To help keep

check on the progress of projects and all the ensuing

documentation, the engineering department has

installed a new Product Data Management system.Specificationreview

Project orderreview

Design/drawing

approval bycustomer

Componentquality control

of selectedcomponents

• Mechanicalinspection• Electricalinspections• Work shopinspections

Factoryacceptance

tests

• Functional tests• Equipmentcalibration

• DocumentationRAB

Installationtests

Columns are usually delivered directly tothe customer after assembly at thesubcontractor. Comprehensive qualitycontrol and inspection are also performedduring assembly.

After delivery, help with installation andoperation of all systems and columns isavailable as well as training for personnelvia our Fast Trak Services.

Column packing studiesIn our testing facilities we also carry outextensive packing studies on columns andmedia. These studies are designed to ensureoptimal performance of media and columntogether as a separating unit, to givesmooth and immediate start-up. Proceduresinclude evaluation of column packing bymeasuring height equivalent to a theoreticalplate (HETP) and asymmetry values. Theknowledge and expertise gained fromextensive testing form the basis of ourcolumn packing recommendations and alsolay the foundations for new productdevelopment, as was the case withFineLINE columns and SOURCE media.Packing studies are performed on allproducts lines. This is an on-going processin our programme to continuously improveproduct performance. For details of ourpacking studies, see Downstream #26.

* Trademark of Haynes International Inc.

RAB = Revised as Built.

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Fast Trak assistanceSupport for columns, media andequipment is also available separatelythrough Fast Trak Services. They canprovide courses, seminars and workshopscovering all aspects of industrialchromatography, help you speed upvalidation by having InstallationQualification (IQ) and OperationalQualification (OQ) documentationavailable for our systems and columns,and offer advice about how to meetregulatory requirements. Fast Trakprovides Validation Support Files forUNICORN control software andcolumns, as well as Regulatory SupportFiles for media. These files containdocumentation such as materialproperties, certificates of analysis,

toxicological studies, Material Safety DataSheets and much more; all designed to aidvalidation and speed up time to market.

Complete solutionsCustom-designed solutions are anenjoyable challenge. Our new and modernengineering facilities have increased ourcapacity, helping us to meet the wideranging and growing number of customerrequests. And although you may becontacting one of our representativesclose to you, the engineering departmentin Uppsala, Fast Trak Services world-wide, or experienced personnel in ourregional centers, all are available toensure that all your needs are fulfilled.Our specialists cross discipline boundariesto provide complete solutions andcomprehensive support.

ISPPP ‘9818th International Symposium of Separation and Analysis ofProteins, Peptides and Polynucleotides1–4 November 1998, Hotel Hilton, Vienna, Austria

The symposium will address issues of separation andanalysis of proteins, peptides, polynucleotides and relatedbiomolecules with modern techniques. It will also discussthe new challenges to protein purification introduced bygenomics and proteomics. Lectures, posters, workshops andexhibits will cover the fundamentals of both chromatographyand electrophoresis of biomolecules, development ofbiological and chemical libraries for affinity ligands,industrial chromatography of biomolecules and validation,PCR and related techniques for analytical and preparativepurposes, the impact of genomics on protein separation,and much more.

Full programme details are available from the SymposiumSecretariat:Come In Congress-Meeting-Incentive OrganisationGabriele ProhazkaAlserstasse 32/3, 1090 Vienna, Austria

Tel: +43 1 402 471412Fax: +43 1 402 471414E-mail: [email protected]

Web site: http://www.boku.ac.at/iam/isppp98/

BIOEUROPE 98Bioseparation and Bioprocessing of Biological Molecules7–9 September 1998, Queens’ College,University of Cambridge, England

This is a three-dayprogramme of lectures byinternationalbiotechnologists andscientists. Lectures will covera range of topics associatedwith the separation andprocessing of therapeutics,including current developmentsin upstream and downstreamprocessing, gene therapy,transgenics, regulatory affairs,process economics, and technology implementation.Further details and a full programme can by obtainedfrom the organiser:

Dr G. Subramanian Tel/Fax: +44 1227 720819 E-mail: [email protected]

Announcement

Testing column and media performance.

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ÄKTAFPLC is the newest additionto the ÄKTAdesign platform ofchromatography systems. This

platform comprises a number of systemsall sharing a common way of working,the common denominator beingUNICORN control and data evaluationsoftware. ÄKTAFPLC is a development ofthe original and innovative FPLC systemon the ÄKTA platform, and is intendedfor lab scale protein purification in themicrogram to milligram range. It uses thelatest version of UNICORN, version 3.0for Windows* NT, is fully automated,biocompatible and can be used with allaqueous chromatographic techniques.Measuring 380x480x470 mm,ÄKTAFPLC is very compact and occupieslittle space on the lab bench. In fact, it’ssmall enough to be placed inside a coldbox or refrigerator.

ReliableThe heart of the system is based onreliable pump technology. Pump P-920 isa development of the P-500 pump used in

In 1982, protein purificationunderwent a revolution with the

introduction of FPLC System,which resulted in drasticallydecreased purification times.Over the years FPLC has been

continuously developed toinclude a complete range of

separation chemistries,automation, UNICORN control

software, improved monitoring,compatibility with modern

chromatography matrices, etc. Itsperformance and reliability havemade it an “industry standard”

with over 50,000 citations in theliterature. Now FPLC takes a leap

on to the ÄKTA platform.

FPLC System. It produces accurate,reproducible, pulse-free flow and precisegradient formation. It is compatible withhigh salt solutions and it can deliver flowrates of up to 20 ml per minute.

Easy to learn and useChromatography is a tool used by avariety of scientists in a variety ofapplications. As a tool it must be easy tolearn and use. UNICORN, the brains ofthe system, is pre-programmed withmethod templates, knowledge about thecolumns and system set-ups. All the userneeds to do is to select the appropriatetechniques, template, column and systemset-up, then press run. There is even abuilt-in Adviser ready to help whenneeded. ÄKTAFPLC is extremely userfriendly and exceptionally knowledgeable.As much expertise as possible has beenpacked into the system so that you don’tneed to be an expert chromatographer touse it, but you will get expert help.

* Trademark of Microsoft Corp.

ÄKTAFPLC –the new standardin proteinpurification

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Convenient and flexibleThe system is small and compact, yet itcan accommodate a number of extravalves, sample pumps, air sensors andcolumns for different systemconfigurations to meet the needs ofdifferent applications and differentlaboratories. These accessories are simplyclipped on to the gate rack. The gate rack

can be opened if more space is required,or extended if longer columns (1 m)

are used.

New UNICORN forWindows NT This new version of

UNICORN, v. 3.0 for WindowsNT is designed specifically tocomply with GLP/GMPrequirements and to meetcustomer demands fornetworking and multi-tasking.The improvement in networking

allows you to check the progressof a separation from any other

UNICORN workstation in the network.Remote surveillance possibility means thatyou can avoid having to sit in the coldroom with your equipment and that youcan follow progress in several separationsin parallel.

UNICORN is common to all ÄKTAdesign

systems and provides a consistent way ofworking. The user interface is the samewhether the application is a routine MAbpreparation, purification of a pioneeringnew receptor, or a large-scale downstreamprocess. This consistency reduces trainingtime, the risk of introducing errors andfacilitates scale-up/transfer of methodsfrom the lab bench to the production hall.

Application areas for ÄKTAFPLCIn our own laboratories and incollaborations with experts from researchgroups, ÄKTAFPLC has been tested inseveral applications involvingcharacterisation studies, structure andfunction studies, and optimisation ofmethods. The following are brief

summaries intended to give you an idea ofthe wide-ranging potential of ÄKTAFPLC.Full details are given in the respectiveApplication Notes.

Characterisation studies

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Column: Mono Q HR 5/5Binding buffer: 20 mM Tris*-HCl, pH 7.5,

with 1% octyl-b-d-glucosideElution buffer: 20 mM Tris-HCl, pH 7.5,

with 1% octyl-b-d-glucoside, 1 M NaCl Gradient: 0–50%, 20 column volumesFlow: 1 ml/minTemperature: 5 °CSystem: ÄKTAFPLC Sample loop: Superloop 10 ml

a) Sample: 2.5 ml fraction1-material diluted 10 timeswith eluent A

b) Sample: 3 ml fraction1-material diluted 10times with eluent A

Lane 1: Low Molecular WeightCalibration kit (LMW)14 400, 20 100, 30 000,43 000, 67 000, 94 000

Lane 2: Detergent extractof Escherichia colimembranes

Lane 3: Flow-through material

Lane 4: Fraction 1 from HiTrapChelating 1 ml

Lane 5: Fraction 2 from HiTrapChelating 1 ml

Fig. 1. SDS electrophoresis on PhastSystem using PhastGel

8–25%, silver staining.

Fig. 2. Chromatographic characterisation on Mono Q HR 5/5. Fraction 1 (a) eluted in one main peak, fraction 2 (b) gave a single

sharp peak.

1 2 3 4 5

The gate rack opens out for

snap-on attachment of columns,

valves and other accessories.Pump P-920 is a twin

reciprocating, syringe pump,

producing accurate, pulse-free

flow-rates.

Monitor UPC-900 can detect UV, as

well as monitor conductivity and pH.

It also has automatic scaling of the

UV, conductivity and pH signals.

In one collaboration, ÄKTAFPLC wasused to isolate a integral human cellmembrane protein expressed in E. coli.

When purity demands are very high,purity should be assessed bycharacterisation of the size and charge ofthe protein to avoid erroneousconclusions from furtherstructure/function studies, for examplewith SDS PAGE. The target protein was arecombinant histidine-tagged cytochromebo3 ubiquinol oxidase expressed in E. coli.

Capture was performed on a HiTrapChelating column at cold roomtemperatures in the presence of non-ionicdetergents. The collected fractions wereanalysed by SDS-PAGE for purity (Fig. 1).Under non-denaturing conditions, anionexchange chromatography on Mono QHR 5/5 (Fig. 2a, b) confirmed the highpurity and charge homogeneity observedwith SDS-PAGE. Gel filtration onSuperdex 200 HR 10/30 size gaveessentially one peak, indicating anhomogeneous monomeric protein.

* Trademark of Rohm & Haas Inc.

For more information about ÄKTAFPLC, ÄKTAdesign chromatography systemsor any of the applications work reported here, ask for:

Data Files:ÄKTAFPLC Code no 18-1128-41ÄKTAexplorer System series Code no 18-1124-09ÄKTApurifier Code no 18-1119-48Monitor UPC-900 Code no 18-1128-40UNICORN control system Code no 18-1111-20

Application notes:Rapid optimisation and developmentof an automated two-step purificationprocedure for monoclonal IgGantibodies Code no. 18-1128-93Purification and chromatographiccharacterisation of an integralmembrane protein Code no. 18-1128-92Purification of a labile, oxygensensitive enzyme for crystallisationand 3D structure determination Code no 18-1128-91

See also faxback, page 25

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CaptureColumn: HiPrep 16/10 Q XLSample: Clarified E. coli extractSample volume: 40 mlBuffer A: 50 mM Tris-HCl, 1 mM EDTA,

pH 7.5; 2 mM DTT, 0.2 M benzamidine-HCl, 0.2 mM PMSF

Buffer B: A + 1.0 M NaClGradient: 0% B in 5 CV, 30% B in 5 CV,

100% B in 5 CV (step gradient)Flow: 10 ml/min (300 cm/h)Methodtemplate: Modified basic_gr

Intermediate purificationColumn: SOURCE 15ISO, packed in HR 16/10 column

(16 mm × 50 mm bed) Sample: DAOCS pool from HiPrep 16/10 Q XLSample volume: 40 mlBuffer A: 1.6 M ammonium sulphate, 10% glycerol,

50 mM Tris-HCl, 1 mM EDTA, 2 mM DTT, 0.2 mM benzamidine-HCl, 0.2 mM PMSF, pH 7.5

Buffer B: 50 mM Tris-HCl, 10% glycerol, 1 mM EDTA,2 mM DTT, 0.2 mM benzamidine-HCl, 0.2 mM PMSF, pH 7.5

Gradient: 0–16% B in 4 CV, 16–24% B in 8 CV, 24–35% B in4 CV, 100% B in 4 CV

Flow: 5 ml/min (150 cm/h)Method template: Modified basic_gr

PolishingColumn: HiLoad 16/60 Superdex 75 prep gradeSample: Concentrated DAOCS pool from SOURCE 15ISOSample volume: 3 mlBuffer: 100 mM Tris-HCl, 1 mM EDTA,

2 mM DTT, 0.2 mM benzamidine-HCl, 0.2 mM PMSF,pH 7.5

Flow: 1 ml/min (30 cm/h)Method template: basic_gf

Structure and function studies

Isolation of highly labile proteins in anactive form is a well known obstacle inprotein studies, particularly in structuralbiology where only an active, homogenouspreparation is likely to give crystalsadequate for X-ray diffraction studies.

A purification scheme was developed fordeacetoxycephalosporin C synthase(DAOCS), an oxygen-sensitive enzyme

involved in cephalosporin/cephamycinbiosynthesis. The gene was over-expressedin soluble form in the cytoplasm ofEscherichia coli. The method wasdeveloped by automated screening ofdifferent chromatography media, andgradient optimisation, which wasfacilitated by pre-programmed methodtemplates. The final method involvedcapture on the anion exchangerQ Sepharose XL (Fig. 3a), hydrophobic

interaction chromatography with SOURCE15ISO (Fig. 3b) and polishing with gelfiltration on Superdex 75 prep grade (Fig.3c). The procedure yielded 10 mg ofhomogenous, biologically active DAOCSwhich was used for crystallisation andsubsequent 3D structure determination.The total purification time from cellharvest to collection of DAOCS fromfractions from gel filtration was 5.7 hours,of which the time on ÄKTAFPLCconstituted less than 50%.

Fig. 3a, b, c. The complete, optimised purification scheme for DAOCS using ÄKTAFPLC. The elution positions of DAOCS are shown (Shaded). a)Capture using AIEX. b) Intermediate purification

using HIC. c) Polishing using gel filtration. The true gradient (conductivity trace; red) is shown in a) and b).

a) b)c)

Fast optimisation

In a third study, the flexible design andfunctions of the system were found to beextremely helpful for rapid optimisationof parameters important for the capturestep in a purification procedure and forcreating a routine automated process fortwo-step purification of mousemonoclonal IgG from culture supernatant.

As usual, the aim of the capture step wasto concentrate the target molecule andremove the bulk of the contaminants.Affinity chromatography on HiTraprProtein A was chosen because of its highselectivity for IgG. A pre-programmedmethod template in UNICORN was usedfor automatic scouting of bindingconditions, allowing automatic control ofthe salt concentration of the bindingbuffer. The scouting runs revealed that 2.5M NaCl was optimal for binding (Fig. 4),and was used during subsequent methodoptimisation.

The optimal pH for elution wasdetermined by eluting the antibodies witha decreasing pH gradient ranging frompH 7.4 to pH 3 (Fig. 5). The resultsdemonstrated that the peak containing theantibodies was eluted at pH 4.5, whichwas selected as elution pH in the finalmethod.

Polishing, the final step, was performedby gel filtration on HiLoad 16/60Superdex prep grade column. Thepolishing template is already available inUNICORN.

The two-step procedure was thenautomated by expanding the system withadditional valves and modifying thetemplate according to the newconfiguration. The function MethodQueue in UNICORN enabled the twosteps to be run automatically one after theother. This optimised procedure gave1.2 mg MAb from approx. 50 ml cellculture supernatant. Recovery from bothsteps was above 95%.

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3.0

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Fig. 5. Automatic scouting of optimal elution pH on HiTrap rProtein A. Hydrogen phosphate(pKa 7.2) and sodium citrate (pKa 4.76) were chosen as buffer components.

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Sample: Cell culture supernatant containingmonoclonal IgG1,90 ml

Column: HiTrap rProtein A, 1 mlBinding buffer: 100 mM sodium phosphate,

100 mM sodium citrate,2.5 M sodium chloride, pH 7.4

Elution buffer: 100 mM sodium phosphate,100 mM sodium citrate, pH-gradient from 7.4 to 3.0

Flow: 1 ml/minSystem: ÄKTAFPLC

Fig. 4. Determination of optimal salt concentration in the binding buffer on HiTrap rProtein A.The best binding conditions were defined as those resulting in largest elution peak areascombined with absence of antibodies in the flow-through.

ÄKTAFPLC and proteomicsWith a sound history and a strong lineage,ÄKTAFPLC - designed to handle biomolecules fromnative and recombinant sources – has timed itsentrance well. With the focus today on proteomicsand the vast number of proteins emerging from thehuman genome project, ÄKTAFPLC is sure to quicklyestablish itself as the new standard in proteinpurification.

Sample: Cell culture supernatant containingmonoclonal IgG1, 90 ml

Column: HiTrap rProtein A, 1 mlBinding buffer: 100 mM sodium phosphate,

0-3.5 M sodium chloride, pH 7.4Elution buffer: 100 mM sodium citrate, pH 3Flow: 1 ml/minSystem: ÄKTAFPLC

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STREAMLINE expanded bedadsorption brings upstream anddownstream processing closer by

enabling direct capture of biomoleculesfrom the fermentor. Growing interest inthis new unit operation has called for abroader range of column dimensions. Theintroduction of STREAMLINE 100 is aresponse to this request. TheSTREAMLINE family now boasts a rangeof columns with standard diameters of25, 50, 100 and 200 mm for methoddevelopment to pilot scale and small-scaleproduction. Larger diameters for full-scalemanufacturing operations are available ascustom-built columns. The addition ofthis new dimension is another step in thescalability of STREAMLINE.

Ideal for pilot-scaleSTREAMLINE 100 column is ideal forpilot-scale applications, typically handlingfermentor volumes from 5 to 100 litres atflow rates between 16 and 31 litres perhour. The column is a 950 cm long glasscylinder with an inner diameter of100 cm. The column is easy to operate asthe adaptor is moved hydraulically; thisallows for elution in packed bed mode ifrequired. Recommended settled bedheights lie between 10 and 30 cm,corresponding to adsorbent volumes of800 ml to 2 litres. STREAMLINE 100 issuitable for use with all STREAMLINEadsorbents.

Arrests target moleculeIn expanded bed adsorption withSTREAMLINE, biomolecules such asproteins are captured directly from crudefeedstocks in a single pass operation,

Growing interest in STREAMLINEcolumns for method optimisationand for pilot-scale production has

encouraged us to increase therange of dimensions. With the

introduction of STREAMLINE 100,there is now a column for

capturing product from fermentorvolumes up to 100 litres.

STREAMLINE 100 –mid-range expansion

without the need for any prior dilution orclarification. The principle of operation iscommon to all STREAMLINEdimensions. In the bottom of the columnis a unique liquid distributor. When bufferis pumped through column, the settledadsorbent beads, which have a uniformdensity distribution, are lifted in such away that they form a stable expandedbed. Flow of the target molecule throughthe column is arrested by the expandedadsorbent, while particles and otherdebris flow freely through the column.The target molecule is then eluted fromthe column in packed bed mode, partiallypurified.

Hygienic designLike all STREAMLINE columns, this newdimension is designed for use inenvironments which demand highstandards of hygiene. Materials ofmanufacture include borosilicate glass,electropolished stainless steel,polypropylene and EPDM rubber, all ofwhich are compatible with the solventscommonly used in biopharmaceuticalproduction. Sanitisation studiesperformed on STREAMLINE 200 showthat the column design is sanitisable with0.5 M sodium hydroxide.

This new column can be operated with amanual or automatic STREAMLINEsystem, or as a stand-alone unit. Itconforms with all EU Machine Directivesand is CE marked. For furtherinformation about this or otherSTREAMLINE columns please ask for theData Files or contact your localrepresentative.

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Suggested materials:Unclarified feedSTREAMLINE 25 columnSTREAMLINE adsorbent, 50–150 mlFermentor volume, 0.25–5 litres

Scale up to pilot scale

Verification of the optimised methods forthe final process can be carried out byscaling up to STREAMLINE 50, 100 or200.

Suggested materials:Unclarified feedSTREAMLINE 50 columnSTREAMLINE adsorbent, 200–600 mlFermentor volume, 1–20 litres

Unclarified feedSTREAMLINE 100 columnSTREAMLINE adsorbent, 0.8–2 litresFermentor volume, 5–100 litres

Unclarified feedSTREAMLINE 200 columnSTREAMLINE adsorbent, 3–9 litres Fermentor volume, 20–300 litres

Recovery of Biological Products IXMay 23-28 1999, Whistler, British Columbia, Canada

This conference has established itself over the years as the forum fordiscussion and development of new areas in the exploration andcommercialisation of biological products, especially withinbioseparations and bioprocessing. The established format of morningand evening sessions will be followed, with emphasis on both oral andposter presentations by academic and industrial experts on separationscience and related fields. Key themes will be “Speed in Development”and “Decision Making During Process Development”.

For more information contact:Joan SaluzziRhema Association ManagementPO Box 411106 Tel: 415 487 9857Fax: 415 487 9803 E-mail: [email protected]

Web site: http:// www.novo.dk/R9/

Announcement

Data Files

STREAMLINE columnsSTREAMLINE 50, 100 and 200

Code no. 18-1127-35

STREAMLINE 25

Code no. 18-1112-02

STREAMLINE adsorbents

STREAMLINE SP and DEAE

Code no 18-1111-73

STREAMLINE rProtein A

Code no. 18 1115-67

STREAMLINE Q XL and SP XL

Code no. 18-1123-81

STREAMLINE chelating

(ask for details)

Application Notes

STREAMLINE Scale up

Code no. 18-1118-68

STREAMLINE SP and DEAE

Cleaning-in-Place

Code no 18-1115-27

Process development with STREAMLINE columnsMethod scouting with aconventional packed bed

Selection ofSTREAMLINE adsorbentand determination ofoptimal conditions forbinding and elution arebest achieved throughscreening of clarified feedin a small packed bedcolumn

Suggested materials:Clarified feedStandard column XK 16/20STREAMLINE adsorbent, 20–30 mlOptimisation at laboratory scale

Optimisation in expanded bed mode

The search for optimal throughput, sampleload, dilution factors, flow rates during sampleapplication and elution, etc is carried out withthe smallest STREAMLINE column diameter,using crude sample and bed expansion.

12


Cancer, atherosclerosis, osteoporosis, arthritis and Alzheimer´sdisease are all examples of disorders characterised by specificchanges in the activities of genes. Even infectious disease usuallyprovokes the activation of identifiable genes in a patient´simmune system. Gene therapy aims to treat such illnesses at themolecular level by delivering DNA to affected cells so that thecells will produce a therapeutic protein or peptide. The genesare usually first put into vectors such as viruses or plasmidscomplexed with lipids able to deposit foreign genes into cells.The production of plasmids and viruses for gene therapy isamong the challenges currently emerging for large-scalepurification technology. This article will describe purificationmethods for plasmid DNA and an adenovirus vector for use ingene therapy.

Purification of plasmid DNA Although a wide range of plasmids are now routinely purified atthe lab bench, the methods used are often based on one-use kits.In addition, the production of plasmid DNA for preclinicaltoxicology human trials, and ultimately for an approvedpharmaceutical indication, requires the development of aprocess that reproducibly meets the quality and regulatorystandards for the manufacture of a biopharmaceutical.

For gene therapy, a plasmid should be in the supercoiled formand essentially free from bacterial chromasomal DNA, RNA,proteins and endotoxins. Target levels for supercoiled plasmidand impurities in plasmid preparations are listed below, (2).

Target levels for supercoiled plasmid and impurities in plasmidpreparations.

Content TargetProtein undetectableSupercoiled plasmid >95% nucleic acidRNA <2% nucleic acidChromosomal DNA <1% plasmidEndotoxin <100 EU/mg plasmid

The common host for plasmid DNA production, as forrecombinant proteins, is E. coli. Plasmids extracted from E. coli

by alkaline lysis are recovered in the clear lysate supernatantobtained after centrifugation. However the plasmid onlyaccounts for a small percentage of the total nucleic acid contentof the lysate (the higher the plasmid copy number, the higher the

One of the new molecular approaches to medicineis gene therapy which aims to correct disease atthe genetic code level. To achieve this, additional

genetic material must be delivered safely andeffectively to target cells. There are several

delivery strategies currently under investigation:one is based on modified viruses, another is basedon liposome complexes, yet a third injects nakedDNA. The large-scale purification of such deliveryvehicles, or vectors as they are known, is one of

the challenges that must be met for gene therapy tobecome a viable clinical tool. Below we describe

the development of two purification strategies, onefor plasmid DNA, the other for a viral vector.

Towards large-scaplasmid DNA and a vir

human gen

Cell paste

Resuspension in buffer

Alkaline lysis

Neutralisation

Addition of CaCl2

Centrifugation

Filtration – glass wool

Filtration 0.45 µm

Capture – Q Sepharose XL

Polishing – SOURCE 15Q

Plasmid for gene therapy

Flow scheme for purifying plasmid DNA for gene therapy.

13


ale purification ofral vector intended forne therapy

concentration), the main contaminants being RNA,chromosomal DNA and endotoxins. Initial cell disruption andclarification steps will affect the release and size ofcontaminating nucleic acid into the starting material forpurification. To obtain the best results from subsequentchromatography steps it is therefore important to use a plasmidwith a high copy number and to use a cell lysis method which isas gentle as possible. Once this has been done the key to asuccessful purification lies in the selective removal of the nucleicacid impurities. In this application plasmid of gene therapypurity grade was purified in three main steps. The methodavoids the use of RNAse thus simplifiying the validationprocess.

Sample preparation

Frozen E. coli cells harbouring 4.5 kb plasmids were used andcell lysis was performed essentially as described by Sambrook et

al (1).

Calcium chloride (0.2 M) was chosen as a precipitant to reducethe amount of RNA, chromosomal DNA and open circularplasmids in the clear lysate. RNA and chromosomal DNA werereduced by about 50% and 70% respectively. About 10% of theplasmid is lost during this step. The precipitation was integratedwith the alkaline lysis procedure, and hence no additionalhandling step was required. The conductivity was adjusted bydilution, where necessary, and the clarified lysate at pH 5.5 wasthen applied directly to the gel adsorbent without any change ofbuffer.

Chromatography

Since RNA and DNA have similar charge properties it has beendifficult to capture plasmid DNA selectively on anionexchangers. In this application 99.4% pure supercoiled plasmidwas obtained in a two-step, rapid and scalable chromatographyprocedure using Q Sepharose XL for capture and SOURCE 15Qfor polishing. Operating conditions were chosen to captureplasmid selectively from the pre-treated lysate.

Vectors are the vehicles being developed for delivering genetic material to the target cell in gene therapy. Vectorsbased on viruses can be effective vehicles for gene transfer due to their specialised mechanisms for binding tospecific types of cells and delivering their payload. Viral vectors containing therapeutic genes, can beadministered directly to patients, in vivo, or used to treat cells in vitro before being administered to the patient.

Viruses presently being developed for vectors for both in vivo and in vitro use include retroviruses, adenoviruses,herpes simplex viruses and adeno-associated viruses. Each type of viral system offers both advantages anddisadvantages with respect to efficiency and accuracy of delivery, contra potential infection. For example,retroviruses integrate copies of their genes permanently into the chromosomes of the cells they invade, offeringlong term stability; however they lack specificity for host cells and can deliver their genes into a variety of celltypes. Adenoviruses are considered relatively safe since they cause nothing more serious than the common cold;however, the body has a strong immune reponse against them.

There are other vector systems under development that do not use viruses, for example those based on liposomesand DNA conjugates. Liposomes can be designed to carry plasmids – flexible loops of DNA that multiplynaturally in bacteria – in which original genes have been replaced with therapeutic ones. Liposomes are nonpathogenic, but are less effective at transferring the genetic material into the cells.

Other studies are focused on non vector delivery, such as direct injection of naked DNA into the patient. DNAcan be taken up and expressed by some cells, but stable transformation is rare.

In all cases, research is focused on enhancing the benefits and negating the drawbacks of each of type of deliverysystem so that safe and efficient delivery of the gene to its target cell is achievable.

14


Fig. 4. The capture step. Chromatographic procedure and conditions.

CV = column volume, X = volume of the clear lysate, Flow velocity is 60 cm/h during the

elution of plasmid and 300 cm/h in all the other steps.

Time

Q Sepharose XL SOURCE 15QBed volume 10 ml 6 ml

Amount of plasmidapplied (mg) 6.2 4.6Analysis of plasmid peakTotal nucleic acid (mg) 5.4 3.8Plasmid amount (mg) 4.8 3.9Plasmid (% nucleic acid) 90 99.4RNA (% nucleic acid) 4 not detectedChromosomal DNA(% nucleic acid) 2 0.6Supercoiled plasmid(% plasmid) 93 100

Yield per step(% of plasmid applied) 78 85Overall yield(% of total plasmid) 78 66

Table 1. Analysis of plasmid fractions from capture and polishing (Figs. 3 and 5).

Capture

Q Sepharose XL was found to show the highest bindingstrength for plasmids of various sizes when compared to severalother ion exchangers (Fig. 1). Method optimisation wasperformed on a 1 ml column (Figs. 2a and 2b) and thepurification then scaled up to a 10 ml column (Fig 3).Conditions used in the capture step are outlined in fig. 4 andanalysis data are shown in Table 1. Note that using a lowerflow velocity during plasmid elution increased the yield by 20%.

Fig. 2a. Purification of plasmid on Q Sepharose XL, 1 ml column.

The procedure is described in figure 4.Column size: 1 ml, 5 mm i.d. x 50 mmSample: 20 ml of clear lysateFractions 2, 3 and 4 were analysed by agarose gel electrophoresis in figure 2b.

Chromatography Sample Elution of Cleaning-process Equilibration application Elution of RNA plasmid in-place

Column volume 5 CV X ml + 5 CV 3 CV 5 CV 5 CV 5 CV

Buffers 0.5 M KAc, 25 mM Tris-HCl, 25 mM Tris-HCl, 50 mM 0.5 M NaOH,pH 5.5 0.75 M NaCl, 0.5 NaCl, Phosphate, 2 M NaCl

1 mM EDTA, 1 mM EDTA, 1 M NaCl,pH 8.0 pH 8.0 pH 10.00

Specialconditions Flow Apply 1.1 CV

velocity Pause–5 min300 cm/h Elution with

60 cm/h for3.5 CV

Column size: 1 ml, 5 mm i.d. x 50 mmBuffer A: 25 mM Tris-Cl, 0.5 M NaCl, 1 mM EDTA, pH 8.0Buffer B: 25 mM Tris-Cl, 1.2 M NaCl, 1 mM EDTA, pH 8.0Sample: 76 µg of 4.5 kb plasmidGradient: 0–60%, 30 CV

SOURCE

30Q

Q Cera

mic Hyp

erD F*

SOURCE

15Q

Q Sep

haro

se Fa

st Flo

wQ S

epha

rose

XL

Cond

uctiv

ity

Fig. 1. Elution of plasmid on different anion exchange media.

Polishing

Final purity goals were achieved by chromatography on a 6 mlprepacked column of SOURCE 15Q (Fig. 5), using a shallowsalt gradient. The content of RNA, chromosomal DNA, andendotoxins was further reduced in this step. The resultantsupercoiled plasmid was essentially free from bacterialchromosomal DNA, RNA, proteins and endotoxins (Table 1)and satisfied the target purity for gene therapy applications(Table 2).

* Trademark of BioSepra Inc.

15


Open circular plasmid

Lane 1: Clear lysateLane 2: Flow through

fraction 2Lane 3: RNA fraction 3Lane 4: Plasmid fraction 4

Supercoiled plasmid

RNA

1 2 3 4

Fig. 2b. Analysis of fractions from Fig. 2a by agarose gel electrophoresis.

The procedure is described in fig. 4. The pooled plasmid fractions are indicated byarrow. The extra peaks during plasmid elution is due to the changes in flow rateduring elution.Sample volume: 810 ml, clarified lysate obtained from 31 g cellsConductivityof sample: 60 mS/cmColumn size: 10 ml, 16 mm ildl x 50 mmThe fractions indicated by arrow were pooled.

Fig. 3. Capture of plasmid on Q Sepharose XL, 10 ml column.

Content TargetSupercoiled plasmid >95% nucleic acidRNA <2% nucleic acidChromosomal DNA <1% plasmidEndotoxins <100% EU/mg plasmid

Table 2. Target levels for supercoiled plasmid and impurities

in plasmid preparations (2).Sample: 140 ml, pooled plasmid fractions from fig. 3Conductivityof sample: 38 mS/cmColumn: RESOURCE 15Q, 6 mlColumn size: 16 mm i.d. x 30 mmGel media: SOURCE 15QBuffer A: 25 mM Tris-HCl, 1 mM EDTA, pH 8.0Buffer B. 25 mM Tris-HCl, 1 mM EDTA, 1.0 M NaCl, pH 8.0Buffer for CIP: 2 M NaCl, 0.5 M NaOHGradient: 40–100%B, 20 CVFlow velocity: 300 cm/hThe fractions indicated by arrow were pooled

Fig. 5. Polishing on SOURCE 15Q.

Cleaning in place

After each run both columns were cleaned in place with sodiumhydroxide and sodium chloride. Tightly bound chromosomalDNA was removed during this step. The column was thenrapidly equilibrated.

Rapid and scalable method

This method is rapid- the two chromatography steps take aboutthree hours and the whole process can be completed within aday. The method is scalable and includes a CIP procedure using0.5 M NaOH and 2 M NaCl.

Comments

This process was optimised using plasmid with a low copynumber. The purification yield is likely to be much higher usingplasmid with a high copy number.

Acknowledgements

The work described here is the result of a collaboration betweenscientists at Rhône-Poulenc Rorer Gencell and AmershamPharmacia Biotech. A patent has been applied for the process.

16


Purification of a viral vector Francis Blanche

Rhône-Poulenc Rorer Gencell

At present, the most efficient and commonly used vectors forgene therapy are based on attenuated or modified versions ofviruses. The modified viruses cannot replicate in the patient, butdo retain the ability to deliver genetic material. Current clinicalprogrammes at Rhône-Poulenc Rorer Gencell utilise adenovirusvectors. A single step purification of Type 5 adenovirus has beendeveloped using SOURCE 15Q media, giving a product whichfully complies with the quality specifications required forclinical grade material (see below).

filtration, ultrafiltrationBenzonase* treatment (optional)

AU

0.40

0.20

0.00

50 100 Time (min)

AU

1.800

1.600

1.400

1.200

1.000

0.800

0.600

0.400

0.200

0.0000.00 5.00 10.00 15.00 20.00 Time (min)

Fig. 6. Preparative scale pruification of adenovirus on SOURCE 15Q.

Fig. 7. HPLC purity check of virus peak from the scaled-up chromatography run.

Vector purification process

culture supernatant293 cells

SOURCE 15Q

diafiltration tofinal buffer

Therapeutic virus

Vector quality goals (3)HPLC purity (see Fig. 7) > 98%SDS-PAGE Coomassie purity Only protein bands corresponding to

constitutive viral proteins detectedBSA < 50 ng per dose1

Host nucleic acids < 10 ng per dose1

Host cell protein None detectable (Western blot)Infectivity Same as CsCl purified1 Current maximum dose = 2.5 x 1012 vp.

Sample preparation

A “supernatant harvest method” was used in which theculture is continued until autolysis of packaging 293 cellsoccurs (patent applied for) (4). After filtration andultrafiltration steps the sample was applied to a columncontaining SOURCE 15Q. Optionally, a treatment with anuclease (Benzonase) can be introduced beforechromatography to provide an additional guarantee as to theremoval of host cell nucleic acid.

One-step purification on SOURCE 15Q

Initial experiments used a 1 ml RESOURCE column on FPLCSystem. Process development studies were done onÄKTAexplorer 100 using FineLINE Pilot 35 column. A saltgradient elution method was developed and optimised onÄKTAexplorer 100. Conditions were optimised to meet set

quality specifications. Note that information about operatingconditions is proprietary. After the chromatography step, thepurified virus was transferred to the final buffer usingdiafiltration.

In this process it was possible to obtain clinical grade materialusing a single chromatography step on SOURCE 15Q. The littlebump which is in the tailing edge of the virus peak has beenidentified as empty particles (devoid of viral DNA). It iscollected as a separate fraction and discarded.

Scale-up

The process was scaled up to 2.5 litres of medium inFineLINE 200 column operated by BioProcess System. Scale upwas done keeping load per volume of SOURCE 15Q, flowvelocity and bed height constant, Fig. 6. Purity of the virus peakwas greater than 98% as determined by HPLC, Fig. 7.

* Trademark of Nycomed Pharma A/S.

17


Cleaning in place

After each run the column was cleaned with sodium chloride,sodium hydroxide and phosphoric acid/sodium chloride.Phosphoric acid was found to be more efficient than acetic acidin removing tightly bound nucleic acids.

At least 20 purification cycles can be run without any reductionin column performance.

Comments

Due to the excellent performance of SOURCE 15Q it waspossible to develop a one-step purification process for theclinical production of adenovirus for gene therapy. Particlerecovery during purification was consistently between 75% and90% irrespective of scale and met the specifications for purity asoutlined in vector quality goals.

Chromatography on SOURCE 15Q also allows for separationof infectious and non-infectious particles. Non-infectiousparticles are undetectable in final preparations.

Residual Benzonase was not detectable in final product(< 0.2 ng / 2.5 x 1012 vp).

The chromatography step takes less than three hours, CIP takesseveral hours and the whole process from harvest to filledproduct takes two days.

Enormous potential These studies are a contribution to the efforts being made andto meeting the challenges in developing molecular medicine.There are still many hurdles to pass, ethical as well as technical,but the potential for gene therapy is a fantastic driving force.

Patents

Rhône-Poulenc Rorer has filed a patent application for theproduction/purification process described above (4).

References1. Sambrook, J., et al. (1982) Molecular cloning: A Laboratory

Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.

2. Marquet, M., et al. (1997) BioPharm June 40–45.3. WHO Expert Committee on Biological Standardization, Forty-ninth

Report. WHO Technical Report Series, WHO Geneva (in press).4. RPR patent application number WO 98/00524.

First Meeting on Plasma Product BiotechnologyMarch 27-30 1999, Daydream Island Conference Centre, Queensland,Australia

This first meeting on Plasma Product Biotechnology will focus onmodern aspects of plasma fractionation technology, such asrecombinant plasma products, and the needs of those involved inplasma product development and production, such as productsafety and process economics.

The programme will consist of oral and poster presentations withplenty of opportunity for informal discussions. Speakers will discusslarge-scale production of albumin, immunoglobulins, coagulationfactors, fibrin sealants, other therapeutic products, as well as pathogen removal and inactivation, and regulatory issues.

In addition, participants will also have the opportunity to visit CSL in Melbourne before the conference. CSL operatesthe world’s largest chromatographic albumin plant.

For more information, please contact the Secretariat at the address below:

PPB 99, SecretariatB.O. ConferenceP.O. Box 10078SE 75010 Uppsala Tel: +46 18 165060 E-mail: [email protected] Fax: +46 18 304074 Web site: http://www.bo-conf.com/ppb99

Announcement

18


The group of diseases collectivelyknown as transmissiblespongiform encephalopathies

(TSEs) include several human diseasessuch as Creutzfeldt-Jakob Disease(CJD),Fatal Familial Insomnia(FFI) andGerstmann-Strassler Syndrome(GSS). Inaddition, there are other TSEs known toinfect animals, with sheep scrapie beingthe best characterised. Until the 1990s,TSEs had not been of great publicconcern. However, recent issuessurrounding the “Mad Cow”phenomenon in the United Kingdom haveincreased public awareness of thesediseases and recent evidence stronglysuggests that bovine spongiformencephalopathy (BSE) may have enteredinto the human population (1, 2).

Although disputed, the causative agent forthese diseases is generally believed to bethe prion protein. In the diseased state,the prion protein is in an alteredconformation that makes it moreproteinase resistant, less soluble and isfrequently used as a marker for disease(for more comprehensive reviews seereferences 3 or 4 ).

Regardless of the nature of infectivity, theTSE agent is tenacious and resistant tostandard methods of pathogeninactivation. Indeed, methods commonlyused to inactivate viral or bacterialpathogens such as autoclaving (5, 6), pH

Methods of inactivation oftransmissible spongiformencephalopathies (TSEs)as applied to productionchromatography processesDouglas Lee*, Wytold Lebing, Christopher Stenland, Stephen Petteway, Jr.Bayer Corp.P.O. Box 507Clayton, NC 27520

extremes (7, 8), detergent treatments (7),high temperatures (9) or evencombinations of these methods (5), do notguarantee complete inactivation of TSEagents.

With recent discussions from theEuropean Union and the FDA regardingthe banning of pharmaceuticalscontaining bovine- or human-derivedproducts, pharmaceutical companiesaround the world are attempting tounderstand and develop procedures forinactivation of TSE agent(s). Sweepingbans of these products could potentiallyinclude not only animal-derived productsbut also recombinant products whichutilize materials obtained from animaltissues, products obtained from cellsgrown in the presence of animal seraand/or products which may use an animalproduct as a stabilising excipient (e.g.albumin). Unfortunately, efforts toinactivate the TSE agent are hindered notonly by the resiliency of the infectiousagent but also the cumbersome andlengthy bioassay required to assessinfectivity.

The purpose of this review is to comparesome of the chemical and physicalmethods that have been reported to affectthe agent and to assess the potentialapplication of these methods to thepharmaceutical industry. It is important toemphasise that while no method has been

shown to totally inactivate the TSE agent,some methods have demonstratedeffective diminution of infectivity. Thedata reported within reflects bioassaydata obtained from animal studies usingsamples treated in idealised systems (e.g.purified or dilute infective material). Forany given process step where TSEinfectivity is to be assessed, infectivityinactivation and/or removal would needto be determined under the specific set ofconditions from which the processsolution is derived.

Finally, conclusions extrapolated frombioassay studies must always take intoaccount the specific TSE strain utilised inthe study. For example, BSE may have adifferent resistance profile to a giveninactivation method compared to aparticular rodent-adapted sheep scrapie.It is always in an investigator’s bestinterest to use the most relevant strain ofTSE and/or the most resilient strain toinsure maximum efficacy against anunknown TSE burden (10).

Chemical methods ofinactivationTable 1 lists several chemical agents whichmay be of utility to inactivate TSEinfectivity. In general, most organicchemicals necessary to quantitativelyinactivate TSEs are harsh on mostpharmaceutical process equipment. For

* Corresponding author. ©“F

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lbee

”/T

he I

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ank.

19


example, acidified aqueous phenol whichhas a high probability of success to killthe agent (8), would be impractical giventhe hazardous nature of the material andequipment corrosion issues that surroundthe use of such a solution on themanufacturing floor. Other phenol andphenol combinations demonstrated someeffectiveness as well (7). Finally, theformaldehyde combinations typically usedto fix tissues for histology purposesgenerally failed to effectively inactivatethe TSE agent (5).

Other possible inactivating chemicalsvaried in their efficacy to reduce TSEtiters. For example, although chaotropicchemicals (guanidine) at higherconcentrations showed some inactivationof the TSE infectivity (7), detergents (SDS,N-lauryl sarcosine) proved to have nosignificant effect (7, 11). Acids at veryhigh concentrations proved to beeffective, but their effectiveness alsodiminished at lower concentrations (7).

Sodium hydroxide, in most cases, is anexcellent sanitising agent for viralpathogens and is compatible with manyGMP cleaning regimes, including manychromatography media. However, in theTSE literature there are somediscrepancies in data reporting itseffectiveness. In general, sodiumhydroxide at 1–2 M concentrations doesdecrease infectivity of some of the TSEagents tested, but in no case that has beenreported has sodium hydroxide (or anyother chemical) totally and consistently

removed infectivity. Indeed, as shown inTable 1, its effectiveness variessignificantly from study to study.

Physical methods of TSEinactivationTable 2 is a brief compilation of variousphysical methods that have been used toinactivate the infectious entity. Althoughnot practical for use directly on processsolutions, it is possible that some of thesetechniques may be used to treatchromatography media and/or processequipment.

Irradiation using g-rays, proved to beineffective on TSEs of human origin (12).Likewise, UV irradiation at varying dosesand wavelengths also proved to beineffective (13). However, it should benoted that many of the irradiationexperiments in the TSE literature aredifficult to interpret given variations indosing parameters such as time, samplesize (mass and volume), effectiveness ofpermeation, etc.

While autoclaving has been demonstratedto insure sterility against viral andbacterial pathogens, the effectiveness ofautoclaving against TSE agents is lesscertain. This observation is complicatedby the number of configurations availableto perform an autoclaving experiment.While no autoclaving technique canguarantee a complete kill of the TSEagent, gravity displacement at 135 °C for1 hour or longer is generally consideredeffective at inactivating over 5 logs ofinfectivity (8).

Boiling infected brain homogenate forshort time periods has proven moderatelyeffective, removing less than 3 logs ofinfectivity (9). Dry heat at temperaturesof 160 °C has shown to remove 6 to7 logs of infectivity (9). Ashing materialsat 360 °C for 1 hour or incineration ofmaterial is also an effective method toinactivate the TSE agent (5) although nota viable option for a process step forpharmaceutical products.

Chromatography resinconsiderationsCleaning of chromatography resins andsupporting process equipment provides anadditional challenge in processdevelopment. For any contaminatedproduct that contacts resin, it is generallyassumed that some residual proteinremains on the resin. For this reason itwould be useful if some level ofinactivation of TSE by either chemical orphysical methods could be employed.

Of the chemicals listed in Table 1, NaOHis probably the most commonly usedcleaning agent in manufacturing processesand most non-silica resins will withstand

up to 2 M NaOH, within certain timeconstraints. Likewise, 7 M Guanidine-HCl, 3 M Guanidine-thiocyanate, andmethanol/sodium hydroxide arepossibilities, but they are more difficult touse at a larger scale. The majority of theother agents listed would either damagethe resins or present handling dangers inthe manufacturing setting.

Sodium hypochlorite may be a practicalalternative to NaOH at themanufacturing scale. In the literature,sodium hypochlorite is described as agood inactivation agent for TSEs ifchlorine concentrations are maintained atsuitable levels (6, 10). If handled properly,at ambient temperature, sodiumhypochlorite is less hazardous and aseffective as heated NaOH. However,process equipment must be modified insuch a manner that it is able to toleratethe corrosive nature of the hypochlorite,and chlorine concentrations should becarefully monitored to insure properlevels of chloride are maintained.

Most of the physical methods listedwould not be practical on themanufacturing floor. For example, forirradiation to be effective it would berequired that adequate and consistentenergy levels are provided to insurecomplete and overall effectiveness of thetechnique. This would be difficult forchromatography resins used in eithercolumn or batch formats. Heating may beuseful, although the extreme conditionsrequired for effective inactivation wouldbe difficult to maintain and validate. Acompromise solution might include acombination of physical (e.g. heating) andchemical (e.g. NaOH) methods. For eitherheating or a combination techniques, theresin must be able to withstand themethod ascribed.

Finally, it should be noted that the resinmanufacturing procedure itself should becertified as TSE free.

Other considerationsAdditional considerations should includethe physical state of the potentiallyinfectious material. For instance, it is

20


Table 1. Comparison of various chemical methods for inactivation of TSE agents. Asterisks (*) indicate that the data was obtained from a graph. Depending upon how the bioassayed was

performed, actual log10 decreases may not be available (NA). Numbers in parentheses indicate increases infectivity. Effectiveness is classified as follows: ≤1.5 log10 decrease, ineffective;

1.5–3.0 moderately effective; ≥3.0 effective. Unless otherwise stated, all assays were performed by rodent bioassay.

Time/ Agent Log ID50Compound Temperature tested change Comment(s) Ref

0.9% Acidified aqueous phenol 0.5 hr/RT1 Hamster Scrapie 5* Effective; compound is aqueous acidic phenol (8)0.9% Acidified aqueous phenol 16 hr/RT Hamster Scrapie ≥7.4 Effective; compound is aqueous acidic phenol (8)9.0% Acidified aqueous phenol 0.5 hr/RT Hamster Scrapie >7.0 Effective; compound is aqueous acidic phenol (8)81% Acidified aqueous phenol 0.5 hr/RT Hamster Scrapie >7.0 Effective; compound is aqueous acidic phenol (8)50% phenol 2 hr/RT Mouse CJD ≥7.0 Effective (7)80% phenol 2 hr/RT Mouse CJD ≥7.0 Effective (7)1 M Guanidine-HCl 2 hr/RT Mouse CJD 0 Ineffective (7)7 M Guanidine-HCl 2 hr/RT Mouse CJD ≥7.0 Effective (7)1 M Guanidine-thiocyanate 2 hr/RT Mouse CJD 0 Ineffective (7)3 M Guanidine-thiocyanate 2 hr/RT Mouse CJD ≥7.0 Effective (7)10% formaldehyde 48 hr/RT Hamster Scrapie 1.5 Ineffective (5)10% formaldehyde/autoclave 48 hr/RT; 0.5 hr/134 °C Hamster Scrapie 1.8 Moderately effective (5)autoclave /10% formaldehyde 0.5 hr/134 °C; 48 hr/RT Hamster Scrapie 6.8 Effective (5)10% formaldehyde 48 hr/RT Hamster Scrapie (0.3) Ineffective (5)15% formalin 96 hr/RT CJD 0 Ineffective; typical tissue fixation technique (14)sat’d phenol-formalin, formalin 24 hr, 72 hr/RT CJD 5 Effective (14)sat’d phenol-formalin,96%formic acid 24 hr, 72 hr/RT CJD >6 Effective (14)15% formalin, 96% formic acid 24 hr, 1 hr /RT CJD >8 Effective (14)1 N hydrochloric acid 2 hr/RT Mouse CJD 0 Ineffective (7)20% formic acid 2 hr/RT Mouse CJD 0.1 Ineffective (7)60% formic acid 2 hr/RT Mouse CJD ≥7.0 Effective (7)80% formic acid 2 hr/RT Mouse CJD ≥7.0 Effective (7)0.2 M trichloroacetic acid 2 hr/RT Mouse CJD 0 Ineffective (7)3.0 M trichloroacetic acid 2 hr/RT Mouse CJD ≥7.0 Effective (7)Hypochlorite (1000 ppm) 0.5–14 hr/RT Mouse scrapie 3.8–≥5.9 Effective at longer incubation times

and dependent on TSE stain (10)Hypochlorite (10,000 ppm) 0.5–14 hr/RT Mouse scrapie ≥4.6 Effective (10)Sodium hypochlorite(8000–16500 ppm chlorine) 0.5–2 hr/RT BSE NA Effective (6)Sodium dichloroisocyanurate(8000–16500 ppm chlorine) 0.5–2 hr/RT BSE NA Moderately effective (6)1 N sodium hydroxide 1 hr/RT Hamster Scrapie ≥5.5 Effective (10)2 N sodium hydroxide 2 hr/RT Hamster Scrapie ≥5.1 Effective (10)0.09 N sodium hydroxide 2 hr/RT/121 °C/1 hr Hamster Scrapie ≥7.4 Effective (8)0.9 N sodium hydroxide 2 hr/RT/121 °C/1 hr Hamster Scrapie ≥7.4 Effective (8)1 N sodium hydroxide 1 hr/RT Hamster Scrapie 6.0 Effective (15)0.25 N sodium hydroxide 2 hr/RT Mouse CJD ~1 Ineffective (7)1 N sodium hydroxide 2 hr/RT Mouse CJD 2.9 Moderately effective (7)2 N sodium hydroxide 2 hr/RT Mouse CJD 1.8 Moderately effective (7)methanol/sodium hydroxide 4 hr/40 °C Hamster Scrapie ≥7.8 Effective (15)methylene chloride/methanol/sodium hydroxide 3 hr/31–33 °C Hamster Scrapie ≥7.8 Effective (15)0.1% N-lauryl sarcosine Added to bioassay

diluent Mouse Scrapie (2) Ineffective (11)1%, 3% SDS 2 hr/RT Mouse CJD 0,0 Ineffective (7)3% SDS 2 hr/60 °C Mouse CJD 0 Ineffective (7)3% SDS 2 hr/100 °C Mouse CJD 0 Ineffective (7)50% ethanol Overnight/4 °C Mouse Scrapie NA Ineffective (16)0.2%-1.0% b-propiolactone 2 hr/37 °C Mouse Scrapie 0.6–1.9 Ineffective (17)ether extraction (equal volume) 10 min/6 °C Mouse Scrapie ~2 Ineffective (18)chloroform extraction(equal volume) 10 min/6 °C Mouse Scrapie 0.7 Ineffective (18)flourocarbon (Freon 113) 10 min/4–6 °C Mouse Scrapie 0.6 Ineffective (18)

1 Room temperature

21


generally contended that if a TSE agentever “dries” onto a given surface, then itbecomes more difficult to inactivate.Therefore, it is recommended that anygiven surface that is possibly infectiousnot be allowed to stand in a dried stateuntil thoroughly cleaned, preferably usingGMP protocols.

Selection of the TSE agent is important inthe evaluation of any of these treatments.Different forms of these diseases mayhave varying degrees of resistance tocertain inactivation treatments (10). Sinceinactivation experiments typically do notshow a linear response to any giveninactivation procedure, a heterogeneityprobably exists within any givenpopulation of infectivity that coulddisplay varying degrees of resistance.

This would imply that some methodswould readily inactivate a certain, specificamount of infectivity resulting in aresidual amount of infectivity that isunaffected by the treatment.

It is important to consider that themajority of the experiments in theliterature represent an idealised situationfor the study of the inactivation of thepathogen. In other words, theseexperiments are typically performed usingsimple brain homogenates or the purifiedpathogen in a simple system. The resultsobtained from these types of experimentsmay be different if other proteins arepresent (e.g. those in a process solution)which may stabilise the infectious agent,thus reducing the effectiveness of thetreatment. Therefore, any given claims ofTSE removal/inactivation would requiresome form of validation/verification.

ConclusionsWe have compiled many of the reagentsand processes that have been tested toascertain any reduction and/or removal ofTSE infectivity. Although, mostchromatography resins cannot withstandmany of the treatments listed, some arecertainly feasible if not completelypractical. Collaborations between bothresin and pharmaceutical manufacturersare required to determine the mosteffective as well as the most practicalmethods to achieve this goal.

Table 2. Physical methods of TSE inactivation. Porous loading (PL) and gravity displacement (GD) autoclaving conditions are indicated. Asterisks (*) indicate that the data was

obtained from a graph. Depending upon how the bioassayed was performed, actual log10 decreases may not be available (NA). Effectiveness is classified as follows: ≤1.5 log10

decrease, ineffective; 1.5-3.0 moderately effective; ≥3.0 effective. Unless otherwise stated, all assays were performed by rodent bioassay.

Duration/ Agent Log Method Conditions Tested Decrease Comments Ref

g-irradiation times varied/doses varied Kuru, CJD NA Ineffective, assayed in primates,much variability was observed. (12)

237 nm UV-irradiation 2 °C Sheep Scrapie 1.8-4.1 Effective at the higher dose tested(2.9x105 ergs/mm2) (13)

250 nm UV-irradiation 2 °C Sheep Scrapie 0.8-1.9 Moderately effective at the higherdose tested (6.5x105 ergs/mm2) (13)

254 nm UV-irradiation 2 °C Sheep Scrapie 1.1-3.1 Moderately effective at the higherdose tested (14.3x105 ergs/mm2) (13)

280 nm UV-irradiation 2 °C Sheep Scrapie 1.1-2.3 Moderately effective at the higherdose tested (106 ergs/mm2) (13)

autoclaving 0.5 hr/134 °C, GD Hamster Scrapie 5.3 Effective (5)autoclaving 0.5 hr/121 °C, PL Hamster Scrapie 3.8 Moderately effective (19)autoclaving 18 min or 30 min/134 °C, PL Hamster Scrapie 7 Effective (6)autoclaving 18 min/134-138 °C, PL BSE 2.5 Moderately effective, however low starting

titer of spiking material (103.6 ID50/g),assayed in rodents (6)

autoclaving 1 hr/121 °C, GD Hamster Scrapie 5.4* Effective (8)autoclaving 1.5 hr/121 °C, GD Hamster Scrapie 5.6* Effective (8)autoclaving 1 hr/135 °C, GD Hamster Scrapie 6.6* Effective (8)autoclaving 1.5 hr/135 °C, GD Hamster Scrapie ≥7.4 Effective (8)heat 10-60 min/70 °C Sheep Scrapie <1 Ineffective, samples were heated

as a homogenate in a water bath. (9)heat 10-60 min/85 °C Sheep Scrapie <1 Ineffective, samples were heated

as a homogenate in a water bath. (9)heat 10-60 min/100 °C Sheep Scrapie 1.6-2.8 Moderately effective, samples were

heated as a homogenate in a water bath. (9)heat 10 min/124 °C Sheep Scrapie 4.3 Effective, samples were heated

as a homogenate in a water bath. (9)dry heat 10,60 min/160 °C Hamster Scrapie 6, 7 Effective, however, there was ≥3.6,

≥2.2 log10 remaining in the treatedsamples respectively. (5)

dry heat 60 min/360 °C Hamster Scrapie 8 Effective (5)

22


References1. Bruce, M.E., et al. Transmissions to Mice Indicate That ‘New Variant’ CJD

is Caused by the BSE Agent. Nature (1997) 498–501.

2. Hill, A.F., et al. The Same Prion Strain Causes vCJD and BSE. Nature(1997) 448–450.

3. Prusiner, S. Molecular Biology and Pathogenesis of Prion Diseases. Trendsin Biochemical Sciences (1996) 455–501.

4. Gabizon, R. and Taraboulos, A. Of Mice and (Mad) Cows – TransgenicMice Help To Understand Prions. Trends in Genetics (1997) 264–269.

5. Brown, P., et al. Resistance of Scrapie Infectivity to Steam Autoclaving afterFormaldehyde Fixation and Limited Survival after Ashing at 360 : Practicaland Theoretical Implications. Journal Of Infectious Diseases (1990)467–472.

6. Taylor, D.M., et al. Decontamination Studies with the Agents of BovineSpongiform Encephalopathy and Scrapie. Archives of Virology (1994)313–326.

7. Tateishi, J., et al. Practical Methods for Chemical Inactivation ofCreutzfeldt-Jakob Disease Pathogen. Microbiology Immunology (1991)163–166.

8. Ernst, D.R. and Race, R.E. Comparative Analysis of Scrapie AgentInactivation Methods. Journal of Virological Methods (1993) 193–201.

9. Mould, D.L. The Response in Mice to Heat Treated Scrapie Agent.J. Comp. Path. (1970) 596–600.

10. Kimberlin, R.H., et al. Disinfection Studies with Two Strains of Mouse-Passaged Scrapie Agent. Journal of Neurological Sciences (1983) 355–369.

11. Somerville, R.A. and Carp, R.I. Altered Scrapie Infectivity Estimates byTitration and Incubation Period in the Presence of Detergents. Virology(1983) 2045–2050.

12. Gibbs, C.J., et al. Unusual Resistance to Ionizing Radiation of the Viruses ofKuru, Creutzfeldt-Jakob Disease, and Scrapie. Proceedings of the NationalAcademy of Sciences (1978) 6268–6270.

13. Latarjet, R., et al. Inactivation of the Scrapie Agent by Near MonochromaticUltraviolet Light. Nature (1970) 1341–1343.

14. Brown, P., et al. A Simple and Effective Method for Inactivating VirusInfectivity in Formalin-Fixed Tissue Samples From Patients with Cruetzfeldt-Jakob Disease. Neurology (1990) 887–890.

15. Martino, A.D., et al. Purification of Non-Infectious Ganglioside PreparationFrom Scrapie-Infected Brain Tissue. Archives of Virology (1992) 111–121.

16. Hartley, E.G. Action of Disinfectants on Experimental Mouse Scrapie.Nature (1967) 11–35.

17. Haig, D.A. and Clarke, M.C. The Effect Of B-Propiolactone on the ScrapieAgent. J. Gen. Virol. (1968) 281–283.

18. Lavelle, G.C. Large Fraction of Scrapie Virus Resistant to Lipid Solvents.P.S.E.B.M (1972) 460–462.

19. Martino, A.D., et al. Inactivation of the Scrapie Agent in a Scaled-DownProcedure for the Purification of Gangliosides from Brain Tissue. Dev.Biological Standards (1993) 187–194.

Conference on affinity chromatography

San Diego, California USA April 16-17, 1998The general theme of this meeting was therethinking of affinity chromatography as acapture step. The advantages of single-steppurification and concentration versus theseeming disadvantages of ligand availability,leakage and cost were discussed in depth.

As a starter for the conference, a workshop onthe current state of affinity chromatographylooked at the possibilities of combinatorialchemistry for production of small syntheticligands aimed at overcoming the availability,regulatory and economic barriers to affinitychromatography used as a first step.

The main part of the conference couldgenerally be divided into two parts: thedevelopment of new affinity chromatographymedia and the purification of monoclonalantibodies using Protein A.

J. Maclennan and T. Ransohoff (Dyax) and L.Varady (ArQule) went over the basics of phagedisplay and combinatorial chemistry forgenerating highly specific ligands.Opportunities for these technologies lie in, notonly affinity chromatography, but also drugdiscovery and gene therapy.

I. Lagerlund (Amersham Pharmacia Biotech)spoke on the importance of having not only anappropriate base matrix but the correctchemistry of attachment (ligand coupling,spacer arms, ligand density and orientation)for coupling these highly specific ligandsgenerated by these new technologies.

A number of case studies were presentedlooking at the process developmentconsiderations for affinity steps used at largescale. Several speakers, including R. Bigelow(Genzyme) showed their work on fluidizedaffinity resins for both recovery and initialpurification.

P-Å. Nygren, (Royal Institute of Technology,Stockholm) showed their work on “Affibodies.”These are ligands made from one of the IgGbinding domains of Protein A which can be re-engineered through phage display methods tobe specific for any target molecule chosen. Heshowed work done on making affinity ligandsfor Taq polymerase and apolipoprotein-A fromthe same starting point.

Presentations on monoclonal antibodypurification using Protein A mainly concernedthe productivity, and the ability to use highflow rates to obtain high productivity usingProtein A for the purification of monoclonalantibodies.

G. Blank (Genentech) gave a clear andstructured presentation of “Scale UpStrategies For Protein A and MonoclonalAntibody Production”, while S. Sommers(Cellex Biosciences) presented results from acomparison study in which the binding ofmurine IgG1 to 7 different IgG affinity resinswas evaluated. The highest binding capacitywas found with rProtein A Sepharose Fast Flowand also the lowest resin cost per mg IgG.

The conference, though small, was focusedand well attended. About 70 attendees werepresent at the conference and 40 at theworkshop.

Meeting report

23


Amersham Pharmacia Biotech willsupply its STREAMLINEtechnology to Yoshitomi, as well as

downstream equipment andchromatography media. According toAmersham Pharmacia Biotech, theventure will likely result in the largestbiopharmaceutical approved usingAmersham Pharmacia Biotech’sSTREAMLINE technology in theproduction process. STREAMLINEexpanded bed technology was chosen forthe initial capture step because it is moreefficient, resulting in faster processingtimes and higher yields when comparedto conventional technology.

“We are pleased to be the leadingsupplier of downstream technology forthis exciting production” said IngvarWiberger, vice president business unitseparations, Amersham PharmaciaBiotech.

The facility, which will be built inChitose, Hokkaido, Japan, will includethe world’s largest STREAMLINE (3x1,000 mm id) and CHROMAFLOW(1,600 mm id) columns to date.Construction will be handled by NiigataEngineering Corp. Ltd., one of Japan’sforemost pharmaceutical industryengineering companies.

Upon completion of the first stage,production capacity will be 12.5 metrictons of pure rHSA per year. Companyofficials expect commercial production of

Uppsala, Sweden (March 23, 1998) – Amersham Pharmacia Biotechand Yoshitomi (formerly Green Cross Corporation) today announcedthat their Bioprocess Group has been selected to provide equipmentfor the world’s first commercial production facility of recombinanthuman serum albumin (rHSA). Due to the risk of cross infection byHIV, or yet unknown viruses, and the associated screening coststhere is enormous pressure worldwide to develop alternatives tousing donor blood to obtain pure human serum albumin (HSA).Clinical uses of HSA include blood volume replacement in cases ofshock, burns and during surgery. Worldwide, HSA is the most widelyused protein for clinical applications.

rHSA, which will be marketed as “Albrec”, to begin in 2000.Plans for the 2nd and 3rd stages are already on the drawing boardwhich will take production capacity initially up to 18 metric tonsand finally up to around 40 metric tons per annum in the newmillennium.

Kazuo Takeuchi, director of the industrial division of AmershamPharmacia Biotech K.K. Tokyo, said, “the choice to useSTREAMLINE over conventional purification technology will helpGCC to maintain their leadership position in the development ofrecombinant human serum albumin.

Amersham PharmaciaBiotech chosen tosupply equipment forrecombinant human serumalbumin production

Press release

First NDAfiling foran antisensedrug

24


The filing of the NDA triggers a significant

milestone payment from CIBA Vision to Isis.

Under the exclusive worldwide distribution

agreement established between Isis and CIBA

Vision in July 1997, Isis will receive $20

million in precommercial fees and milestones.

Results from the Phase III trials of fomivirsen

demonstrated that in both newly-diagnosed

patients with CMV retinitis and in patients

with advanced refractory disease, fomivirsen

produced prolonged delay in disease

progression.

“The filing of the world’s first NDA for an

antisense drug is a seminal event for Isis, our

stockholders and our partner, CIBA Vision,”

said Stanley T. Crooke, M.D., Ph.D., Chairman

and Chief Executive Officer, Isis

Pharmaceuticals. ”We are enthusiastic about

ATLANTA and CARLSBAD, Calif., April 8 /PRNewswire/ —Isis Pharmaceuticals,Inc. (Nasdaq: ISIP) and CIBA Vision Corporation, the eyecare unit of world life sciences leader, Novartis AG,announced today the filing of a New Drug Application(NDA) with the U.S. Food and Drug Administration (FDA)for fomivirsen as a treatment for cytomegalovirus (CMV)retinitis in AIDS patients. CMV retinitis is an opportunisticinfection that results in blindness. Fomivirsen is the firstantisense drug to complete Phase III clinical trialsleading to a regulatory submission.

Isis Pharmaceuticals uses OligoProcess DNA/RNA

Synthesizer and chemicals from Amersham

Pharmacia Biotech in its process for manufacturing

fomivirsen. OligoProcess is a custom-designed,

pump-driven oligonucleotide synthesizer for large-

scale production.

our first commercial opportunity and look

forward to working with CIBA Vision to ensure

a successful launch of fomivirsen in the near-

term.”

“We are very pleased with the Phase III safety

and efficacy results demonstrated by

fomivirsen and believe that the drug, assuming

a favorable FDA review, will be an important

addition to the treatment of CMV retinitis for

patients in need of safe and effective

therapeutic options,” said Daniel L. Kisner,

M.D., President and Chief Operating Officer,

Isis Pharmaceuticals.

“With this filing, we move one step closer to

bringing this new, potentially more effective,

treatment option to AIDS patients with CMV

retinitis,” added Luzi von Bidder, President,

Ophthalmics Business Unit, CIBA Vision

Press release

New book This standard work in the field of protein purification has

been expanded and updated to reflect the state of the

art in the 90’s. Covering a broad range of techniques,

both in chromatography and electrophoresis, it provides

a comprehensive background for anyone moving into the

area in a serious way. Each chapter is written by

different experts which results in a somewhat uneven

level of discussion from technique to technique – some

being more theoretical, others more practical or

applications oriented. Nevertheless, it is hard to find a

more comprehensive single source which covers the

subject in a greater scope or depth.

Worldwide. “Further, fomivirsen is an

important strategic addition to CIBA Vision’s

expanding ophthalmic product portfolio, and

we expect this novel antisense drug to give us

a significant competitive advantage in the

retinal market.”

Protein PurificationEdited by Jan-Christer Janson, Lars Rydén

Second editionPublished by Wiley-Liss, New York, 1998

ISBN: 0-471-18626-0

Amersham Pharmacia Biotech code no.: 18-1128-68

North America

Amersham Pharmacia Biotech Inc.

Attn: Lisa Spangler

800 Centennial Avenue

P:O: Box 1327

Piscataway

NJ 08855 USA

Fax: (800) 267-1352

E:mail: [email protected]

Europe

Amersham Pharmacia Biotech Europe GmbH

Attn: Hans-Rudolf Hoepker

Munzinger Strasse 9

D-79111 Freiburg

Germany

Fax: +49 761 4519 320

E-mai:l [email protected]

Japan

Amersham Pharmacia Biotech KK

Attn: Naoko Imanishi

Sanken Bldg.

3-25-1, Hyakunincho

Shinjuku-ku, Tokyo

169-0073 Japan

Fax +81 (0) 3 5331 9369

E-mail [email protected]

Asia Pacific

K.H. So

15/F, North, Cornwall House

Taikoo Place, 979 King’s Road

Quarry Bay, Hong Kong

Fax: (852)2811 5251

E-mail: [email protected]

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26


Standard courses:VAL 1Validation Issues in Chromatographic Downstream Processing. One and a half dayseminar addressing key issues in validation of chromatographic units.

Course code number: 44-8000-08

DEV 1Process Development: Basic Practical Course on Chromatographic Methods. Threeday hands-on course which gives an overview of chromatographic techniques.


DEV 2Process Development: Practical Course on Chromatographic Methods. Four dayhands-on course in the design and optimisation of a chromatographic process.


DEV 4Process Development: Practical Course on Optimisation and Scaling up to Full-ScaleProduction. Three day hands-on course.


COL 1Packing, Testing and Maintenance of Production ScaleColumns and Standard Operation Procedures.Three day hands-on course.


Customised courses:We can also arrange customised training courses to deal withspecific practical and theoretical topics associated with chromatography anddownstream processing. Customised courses can be given at an Amersham PharmaciaBiotech training center or at your facilities, depending on the equipment involved. Ouraim is to make information and expertise available in the most efficient way for yourcompany.

English is the course language for most of our standard coursesbut we can offer customised courses in French,

German, and Swedish.

1998 Courses atFast Trak Center USACourse datesDEV 1: September 16–18COL 1: September 21–23

1998 Courses atFast Trak Center EuropeCourse dates

DEV 1: November 17–19

DEV 2: November 30–December 4

DEV 4: October 6–8

VAL 1: September 30–October 1

COL 1: November 10–12

For further information about the abovescheduled courses, or validation andconsulting services offered by Fast TrakCenters contact:

USA:Amersham Pharmacia Biotech Inc.

Fast Trak Center

P.O. Box 1327

Piscataway, NJ 08855-1327, USA

Tel: +1 732 457 8064

Fax: + 1 732 457 8246

Europe:Amersham Pharmacia BiotechEurope GmbH

Munzinger Strasse 9

D-79111 Freiburg, Germany

Tel: + 49 (0) 761 4519 152

Fax: + 49 (0) 761 4519 315

Japan:Amersham Pharmacia Biotech K.K.

Sanken Bldg

25-1, Hyakunincho 3-chome

Shinjuku-ku, Tokyo

169-0073, Japan

Tel: +81 (0) 3 5992 3426

Fax: +81 (0) 3 5992 2035

27


International conference on vectorsfor gene therapy: design,production and regulation

March 12-14: Brussels, Belgium

This meeting, jointly sponsored by the European Commission and the FDA was well attended,with nearly 250 participants. The main objectives of the meeting were to discuss key issuesrelated to the scientific development and design of vectors, their clinical-grade production, theiruse in clinical trial, the review process and associated regulations.

Olivier Danos´s (Généthon II & EWGT,CNRS) opening lectureprovided the audience with a concise overview of thechallenges in the development of new vectors for genetherapy. Vectors are experimental agents barely acceptable asdrugs and their efficiency is too low. There is a trend towardsin-vivo gene transfer which creates more problems since theregulatory requirements are more stringent. Tools are notavailable to evaluate all the safety risks.

Jörn Keck (European Commission) argued that the genetherapy industry in Europe is about 5 years behind that in theUS, and of 300 clinical trials, two thirds are in the US. Hesuggested that one reason for this is that the financial climatein the US is more beneficial to start-up biotech firms. Theregulatory situation in Europe and the US was explored byvarious speakers. Harmonisation within Europe still seems tobe some way off.

The subject of intellectual property issues was introduced by lawyer Alex Houtart whodistributed a useful checklist to follow from before the R&D stage to commercialisation. Thepatentability of inventions relating to gene therapy was presented in greater depth byS. Hoekstra from the European Patent Office.

One day was devoted to presentation of basic research on retroviral, adenoviral and other vectorsand control of gene expression.

A solution to the problem of Replication-Competent Adenovirus contamination in the large scaleproduction of recombinant adenoviral vectors was presented by Dinko Valerio (Introgene BV).J. Crouzet (Rhône-Poulenc Rorer Gencell) described the construction of DNA ”minicircles” forimproved transfection efficiency in non-viral gene transfer.

Several speakers described commercial or state-funded facilities for GMP production of vectorsfor gene therapy. The NIH provision of free vector to academics in the US was discussed bydelegates: there are three NIH centres in Michigan, Indiana and Pennsylvania and 35 protocolshave been approved for free vector provision since 1995. There were calls for a similar systemto be set up in Europe to give gene therapy a much-needed boost in the academic sector. Thelast session of the meeting dealt with the validation and testing of vectors with subjects rangingfrom process validation, cell line and vector safety testing to removal of residual DNA in finalproduct and aspects of biosafety in the clinic.

There was also a lively poster session covering a wide span of interests such as vectordevelopment, vector production, cationic lipid delivery, plasmid DNA analysis, vectorbiodistribution analysis and cancer therapy. In addition there were several posters describingmethods for preparative purification of plasmid DNA in expanded and packed beds (TherexysLtd., Instituto Superior Técnico, Portugal/MIT., MAGENTA Corporation, Amersham PharmaciaBiotech -see article on page 12).

Meeting report©

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