A look at contract manufacturing

Transcription

1 STREAMLINE in vaccine process A look at contract manufacturing 40 years of Sephadex Meeting the MAb challenge

2 2 In this issue STREAMLINE in FDA-approved process In Belgium, Smithkline Beecham has recently introduced STREAMLINE TM expanded bed adsorption into one of its production processes for a vaccine ingredient. The vaccine is approved by the FDA and on the market. The 40 th anniversary of Sephadex It is now 40 years since the technique of gel filtration with Sephadex TM was introduced. This article gives a brief history of its development, the resulting product ranges, and related advances in chromatography. New production scale process for human IgG Bayer Corporation, USA, has been investigating means of improving process productivity for Intravenous Immune Globulin G (IGIV). Bayer has developed a novel technique whereby impurities are captured and product flows through the column. Fast Trak Courses New, two-level course on using UNICORN TM control software in an industrial environment; plus the course schedule for spring Contract manufacturing - a way to get things done This is the start of a series of articles addressing contract manufacturing. In this issue we have met Covance Biotechnology Services, USA, and Avecia, UK A talk with Covance Biotechnology Services Re-designed process increases productivity 12-fold Intensification of a large scale commercial monoclonal antibody purification process To meet the escalating demand for MAbs, Centocor BV, The Netherlands, has been able to increase the yield of an existing process by incorporating expanded bed adsorption with STREAMLINE and the novel column packing technique of CHROMAFLOW TM. Collaborations to generate customized ligands Amersham Pharmacia Biotech has entered into agreements with Dyax Corp and ArQule Inc which will give access to phage display and combinatorial libraries respectively, in order to develop novel ligands tailored to customer's application needs. HiPrep 26/10 Desalting columns HiPrep TM 26/10 Desalting is an excellent alternative to dialysis in the laboratory. By coupling four columns in series, 60 ml of sample can be processed in 20 to 30 minutes. Meeting reports 5. Plasma Products Biotechnology Meeting, Queensland, Australia 14. Recovery of Biological Products IX, Whistler, Canada 21. The Fourth Waterside Monoclonal Conference, Norfolk, Virginia, USA 26. The 16 th ESACT Meeting, Lugano, Switzerland Announcements 7. EBA GAb th Interlaken Conference thirty The front cover shows the SOURCE production facility in Uppsala. Further major extensions are ongoing to increase capacity for the growing market. ÄKTA, BioProcess, CHROMAFLOW, Cytodex, Cytoline, Downstream, Fast Trak, FPLC,FineLINE, HiTrap, HiPrep, Sephacryl, Sephadex, Sepharose, Sephasorb, SOURCE, STREAMLINE, Superdex, Superloop, UNICORN are trademarks of Amersham Pharmacia Biotech Limited or its subsidiaries. Amersham is a trademark of Nycomed Amersham plc. Pharmacia and Drop Design are trademarks of Pharmacia & Upjohn Inc. Affinity by Design is a trademark of Dyax Corp. Cytopilot is a trademark of Vogelbusch GmbH INACTINE is a trademark of Pentose Pharmaceuticals Inc. Miraculin is a trademark of BioResources International (BRI) Ghana, Ltd. Nanotiv is a trademark of Pharmacia & Upjohn Inc. Nonafact and Cofact are trademarks of CLB, Sanquin Blood Supply Foundation Tris is a trademark of Union Carbide Chemicals and Plastics Company Inc. Tween is a trademark of ICI Americas Inc. Vigam is a trademark of BioProducts Laboratory Ltd. Windows NT is a trademark of Microsoft Corp. XenoMouse is a trademark of Abgenix Inc. CHROMAFLOW is covered by the following Patents: US 5,213,683; US 5,282,973; GB B; EP STREAMLINE is covered by US Patent 5,522,993 and foreign equivalents. Amersham Pharmacia Biotech AB Björkgatan 30, SE Uppsala Sweden Amersham Pharmacia Biotech UK Limited Amersham Place, Little Chalfont Bucks, England HP7 9NA Amersham Pharmacia Biotech Inc 800 Centennial Avenue, Piscataway, New Jersey 08855, USA Amersham Pharmacia Biotech Europe GmbH Postfach 5480 D Freiburg Germany All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Pharmacia Biotech group which supplies them. A copy of these terms and conditions is available on request. The views expressed by contributors and correspondents are their own and do not necessarily reflect the views of Amersham Pharmacia Biotech Limited. Amersham Pharmacia Biotech UK Limited 1999 All rights reserved Production: RAK Design AB. Printed in Sweden by Westerås Media Tryck AB, November Asia Pacific Tel: Fax: Australasia Tel: Fax: Austria Tel: Fax: Belgium Tel: Fax: Canada Tel: Fax: Central, East, South East Europe Tel: Fax: Denmark Tel: Fax: Finland Tel: Fax: France Tel: Fax: Germany Tel: Fax: Italy Tel: Fax: Japan Tel: Fax: Latin America Tel: Fax: Middle East and Africa Tel: +30 (1) Fax: +30 (1) Netherlands Tel: Fax: Norway Tel: Fax: Portugal Tel: Fax: Russian Federation Tel: +7 (095) , Fax: +7 (095) South East Asia Tel: Fax: Spain Tel: Fax: Sweden Tel: Fax: Switzerland Tel: Fax: UK Tel: Fax: USA Tel: Fax: (9911)

3 thirty 3 SmithKline Beecham, one of the largest vaccine producers, has successfully used STREAMLINE expanded bed adsorption in the purification of a lipoprotein, an active ingredient in a vaccine for human use. The vaccine has been approved by the FDA. STREAMLINE in FDA-approved process BioProcess system and STREAMLINE 400 column used in the purification of a lipoprotein, an active vaccine ingredient. The method developed by SmithKline Beecham uses STREAMLINE TM as an alternative to conventional clarification by centrifugation and filtration during the early downstream steps. In addition to reducing the number of steps, they were able to show that this unit operation was consistent for at least 40 processing cycles. Below, Koen De Heyder, Senior Manager, Scaling Up, describes the development of the capture step using STREAMLINE expanded bed adsorption. CONVENTIONAL process cell broth centrifugation prefiltration 0.22 µm filtration 1 st capture step 2 nd capture step Figure 1. Conventional versus STREAMLINE process. EXPANDED BED process cell broth STREAMLINE 0.22 µm filtration 2 nd capture step The process The vaccine contains a lipoprotein expressed by a genetically engineered Escherichia coli bacterium. Cells are homogenized at high pressure, up to 800 bar, to extract the lipoprotein. Classical processes consist of centrifugation of the cell homogenate to eliminate cell debris followed by a clarification step using filtration or micro-filtration on the supernatant. The supernatant needs to be clarified on a 0.22 µm filter to remove all residual E. coli bacteria. "Centrifugation at industrial scale has not been found to be as effective as at lab scale due to equipment design and a reduced residence time in the rotor," explained Koen De Heyder. "An alternative to the centrifugation and clarification steps at production scale which we considered is the use of expanded bed adsorption chromatography. (See Figure 1.) The cell homogenate can be applied directly onto a STREAMLINE ion exchanger in expanded bed mode. All cells, cell debris, particulates and most host cell proteins pass through the column while the target protein is captured on the ion exchange adsorbent. To elute the protein, the ionic strength of the buffer is increased and flow is reversed to pack the bed; the target lipoprotein can then be eluted as on a conventional ion exchange column. We found the product volume to be reduced by a factor 2 and this eluate can easily be filtered on a 0.22 µm filter," said Koen.

4 4 Further purification is performed by ion exchange chromatography on two Sepharose TM ion exchangers, then a diafiltration step, and finally gel filtration on a Sephacryl TM medium. The purified lipoprotein is sterile filtered on a 0.22 µm membrane and formulated with aluminum salts. Method development and scale up The method using STREAMLINE was developed on a 50 mm diameter column. Ionic strength during capture was lowered by dilution with plain water and the maximum conductivity was fixed based on flow-through analysis. The elution conditions were determined classically as with a conventional ion exchange chromatography column.the adsorbent was washed with buffer containing 1 M NaCl. Cleaning in place (CIP) is performed in expanded bed mode with a NaCl/NaOH solution. The column is stored in 10 mm NaOH until further use. After this capture step, the lipoprotein has been purified 1.5 fold. Endotoxin levels are cleared by a factor of 100, see Table 1. Scale up was performed going from a 50 mm diameter column to a STREAMLINE 400 diameter column. Flow rates were increased in proportion, keeping linear flow velocity constant. Performance of the column was equivalent at both scales. Koen continued, "A problem discovered at the larger scale was the development of precipitates after several purification runs. During cleaning, some DNA particles form which do not pass through the upper net of the column. Solubilization with NaOH/NaCl is not effective, so we opted to work without the upper net. This necessitates accurate control of flow rate through the expanded bed to avoid leakage of adsorbent. In the packed bed, the removal of the upper net did not alter the flow distribution." Table 1. STREAMLINE performance. Volume Total Specific Target Endotoxins (l) Protein (2) activity (1) protein (EU/ml) (g) recovery (%) Starting material STREAMLINE eluate (1) Elisa units/total protein (2) Soluble protein for starting material Validation protein recovery (%) STREAMLINE lifetime validation cycle number thirty CIP of the STREAMLINE column was validated by analysing the equilibration eluate after NaOH/NaCl treatment. The endotoxin level was reduced to < 6 EU/ml whereas product load contains» 10 5 EU/ml for each batch. Column lifetime was tested by running 40 cycles, including CIP, on a small-scale column (25 mm diameter containing 88 ml packed adsorbent). Protein recovery, SDS PAGE profiles of the elution peak, and chromatograms were monitored. Figure 2 shows protein recovery over 40 cycles. The column eluent was tested for endotoxin levels and no increase was detected. In addition, the adsorbent was re-analysed for ionic capacity and no significant change was observed: 200 mmole/ml adsorbent after 40 cycles compared with an initial value of 206. Figure 2. STREAMLINE lifetime validation. Protein recovery of the elution peak is presented up to 40 cycles. It is calculated as the ratio between soluble protein eluted from the column and the total protein content (soluble and not soluble) of the cell broth which is loaded onto the column. The variation in protein recovery is due to variation in the starting material and to the presence of a detergent in the buffer which interferes with the analysis. Powerful alternative "STREAMLINE expanded bed adsorption on crude extract is a powerful alternative to conventional clarification techniques such as centrifugation, micro-filtration and filtration. It clarifies cell broth, concentrates product and purifies at the same time. The lifetime of the column and adsorbents has been proven to be long. Furthermore, the technique can be successfully used in a GMP production," concluded Koen De Heyder. Equipment The chromatography equipment used for downstream processing consists of BioProcess TM Systems controlled by UNICORN TM software, STREAMLINE 400, BPG and a large diameter CHROMAFLOW TM column. SmithKline Beecham is one of the world's largest vaccine producers. It currently has about 20 different vaccines in use around the world. On two sites in Belgium, Rixensart and the newly opened Wavre-Nord, the company has approximately 2500 employees involved in activities related to human vaccines, from research to production and distribution.

5 thirty 5 Plasma Products Biotechnology Meeting Daydream Island, Queensland, Australia Meeting report March 26-31, 1999 These are exciting times for the plasma industry. While established technologies jostle with new technologies, plasma-derived products compete with recombinants, and international regulatory bodies are attempting to harmonize practices, the industry remains united in its common goal - the production of safe and efficacious products. On a tiny island off the coast of Australia, over 100 scientists gathered to discuss critical issues, exchange ideas and foster contacts. The occasion was the first Plasma Products Biotechnology (PPB) meeting, an event which attracted all the major players within the plasma industry and gave ample opportunity for peer interaction and networking. Some of the attendees at the conference. As a prelude to the meeting, jointly sponsored by CSL Ltd, Melbourne, Australia and Amersham Pharmacia Biotech, Uppsala, Sweden, delegates were invited to visit CSL's production plant on the outskirts of Melbourne. These facilities were opened in 1994 and are one of the most advanced for human plasma fractionation, incorporating large scale chromatographic processes in the production of plasma proteins. The CSL plant is today the world's largest producer of chromatographic albumin. The conference took stock of the current situation: the success stories, the not-so-successful stories and future challenges. The plenary lecture by Dr Richard Walker (BioProducts Laboratory, UK) set the scene. He gave an outline of the past and present developments and raised a number of points which may impact the industry in the future; for example, repercussions from the Cochrane report, the possible transmission via blood of new variant Creutzfeldt-Jacob Disease (nvcjd), recombinant products competing with natural products, and the need for harmonization between international regulatory bodies. Seven different aspects of the plasma industry were covered by oral presentations and posters. Albumin The albumin session was particularly lively. Among those present were upholders of the traditional Cohn fractionation method, advocates of the evolving chromatography processing, and producers of recombinant albumin. The safety track record of albumin was clearly stated by Dr Po-Shing Wah (Baxter Immuno-Hyland, USA). The advantages of chromatographic processing were presented by Edward Smith from CSL Ltd, the company which pioneered the use of chromatography in commercial scale manufacturing of albumin. Production of recombinant human serum albumin on a commercial scale was presented by Dr Kaoru Kobayashi (Yoshitomi Pharmaceuticals, Japan). The purification method consists of four chromatography steps, the first of which employs STREAMLINE TM expanded bed adsorption. See also a report in Downstream TM 28. A particularly interesting development was presented by Dr Anky Koenderman (CLB, The Netherlands). It has been discovered that some glycoproteins designed as drug carriers for anti-hiv agents exhibit intrinsic antiviral activity. This activity is related to an increase in negative charge. CLB has subsequently developed a succinylated albumin which shows exciting potential for treating HIV. Coagulation factors Chromatography is widely used in the production of coagulation factors. Since the production of the first Factor VIII concentrates in the sixties, higher purity concentrates have been achieved through the introduction of gel filtration, ion exchange and affinity chromatography, resulting in specific activity of 100 units/mg protein range. Dr Sudhish Chandra (Baxter, USA) went on to describe how this activity was increased further with the implementation of monoclonal antibody immunoaffinity chromatography, a process which yields concentrates with specific activity of 3000 units/mg protein and also inactivates viruses. Other techniques for reducing viruses are the well-documented solvent detergent (S/D) method and nanofiltration. Such techniques are already included in many processes, for example in Pharmacia & Upjohn's (Sweden) process for Nanotiv TM and CLB's process for Nonafact TM, both Factor IX.

6 6 thirty Immunoglobulins Albumin, once the driving force of the plasma industry, has been replaced in this role by intravenous immunoglobulin (IVIG). The demand for IVIG exceeds supply and as a result manufacturers are investing resources into developing more efficient processing methods. One method for fractionation of Factor VIII, von Willebrand Factor, IgG and albumin, developed by HemaSure, Denmark, includes doublevirus inactivation steps, using chromatography and precipitation steps with polyethylene glycol (PEG) and caprylic acid. The purity of the IgG product is greater than 99%, the purity of the other products is greater than 95%. An approach reported by Bayer Corp., USA involves the addition of the salt of caprylate to the IgG suspension before chromatography to removes lipoproteins and inactivate viruses. The chromatographic process is then designed to capture impurities and allow IgG to flow through the column. For details, see page 11. The need for IgG is so great that the market is now demanding the development of stable liquid formulations. The difficulty here, however, is that product stability is a more serious problem than in the lyophilised state. The addition of stabilizers can affect Fc functionality and also induce side effects. Dr John More (BioProducts Laboratory, UK) described the development and characterization of Vigam TM liquid, an intravenous immunoglobulin. Pathogen removal and inactivation Dr Bernard Horowitz (V.I. Technologies, USA) opened this session with a review of the current techniques. The S/D method is well-documented and in widespread use. However there is no universally applicable method. Different methods, often in combination, are currently used. The effectiveness and understanding of the extent to which these different methods inactivate viruses was a topic for CSL. In their process for albumin, the anion exchange step achieved a 3 log reduction for hepatitis C, but only 1.3 log reduction for bovine viral diarrhoea. In the absence of antibody, a log clearance of 4.5 was achieved with hepatitis A virus compared to a log reduction of 1.7 in the presence of antibody. CLB of the Netherlands have introduced a nanofiltration step that combines two nanofilters in series in their production routines for Cofact TM. In trials, this double-filter action was found to completely remove (> 5 log) canine parvo virus and thus increase the safety margins of the final product with respect to transmission of non-enveloped viruses. Dr Horowitz discussed UVC irradiation coupled with other methods. His team has been working with different irradiator designs to achieve maximum virus kill. Dr Joachim Walter (Boehringer Ingelheim Pharma) presented a virus inactivation method employing microwave treatment. Microwave heating using a 5 kw ultratherm showed complete inactivation of enveloped and large non-enveloped viruses at temperatures above 75 C; the non-enveloped virus SV-40 was inactivated to > 4.9 log at 86 C and small non-enveloped viruses like PPV were inactivated by log 2.5 at 97 C. A new technology effective against both enveloped and non-enveloped viruses is INACTINE TM, developed by Pentose Pharmaceuticals Inc. USA. INACTINE acts by triggering an irreversible, covalent modification of nucleic acid, resulting in a non-infectious viral particle. Furthermore, INACTINE does not appear to alter the activity or function of plasma derived proteins. Regulatory perspectives Dr Reinhard Burger (Robert Koch Institute, Germany) described the operation of the German National Advisory Committee on Blood. Comprising a panel of experts from different institutions and associations, the committee's role is to give independent advice to the Ministry of Health. The committee publishes its recommendations on specific topics as "votes" which are accepted by the transfusion community as binding guidelines and are accordingly adopted by blood banks, etc. Professor Rainer Seitz (Paul-Ehrlich Insitut, Germany) presented the current perspectives on recombinant plasma products. Although recombinant proteins eliminate the risks associated with plasma-derived proteins, they are not problem-free. Special controls are needed to ensure the genetic stability of the cell production line, the expression and post-translational modifications. Post-translational processing may cause differences in pharmacokinetic behaviour, as was found with recombinant Factor IX. The development of new technologies such as recombinant techniques and the use of transgenic animals are demanding clearer guidelines from the regulators, and more importantly consensus between international agencies. Quite surprisingly, the regulators requested less paper work!

7 thirty 7 Fibrin sealants Several developments in fibrin sealants were presented, including the addition of antibiotics to deliver efficacious doses over a defined period of time; these include dry fibrin sealant dressings to achieve rapid homeostasis, a spray fibrin sealant foam spray that can control haemorrhage without manual compression, as well as the development of testing methods ex vivo. The future Not so long ago the plasma industry looked to be on shaky ground. Demand for some products had fallen, virus inactivation methods were not efficient and recombinant DNA technologies posed a real threat. Today demand has switched to IgGs, and chromatography is playing a greater role in manufacturing processes by providing better efficiency as well as contributing to virus inactivation and reduction. In addition, specific pathogen removal procedures are being developed and incorporated into manufacturing processes: the combined result of these measures is the production of purer and safer products. A further boost to the plasma industry is the fact that recombinant DNA technologies have proven to be complex and rather expensive. Whilst continuing to focus on improving product quality, the industry is looking to find new indications for existing products as well as new therapeutic products. For example, CLB, The Netherlands, has found evidence that its CI inhibitor for treatment of hereditary angioedema, type 1, is also beneficial for other conditions such as sepsis and myocardial infarction. The Finnish Red Cross Blood Transfusion Service is currently developing a plasma apotransferrin which can be administered intravenously to prevent toxicity resulting from high-dose chemotherapy. Answers in 2001? There are certainly many challenges facing the plasma industry and many questions spring to mind: Will established products find important new indications? Will chromatography extend its role? Will recombinant products impact further on traditional plasma-product markets? Will the regulatory bodies come together, or will their increasingly stringent demands squeeze profit margins and cause some companies to go out of business and others to divest interests. And what of those rogues, the prions, nvcjd, and as yet unknown viruses? You may find some of the answers at PPB in A special edition of Downstream containing synopses of many of the presentations will be available during Please order by code number if you are not already on the mailing list and wish to receive a copy. EBA 2000 The Third International Conference on Expanded Bed Adsorption Garmisch-Partenkirchen, Germany May 14-17, 2000 Expanded Bed Adsorption for the Capture of biomolecules directly from unclarified feed stocks is becoming an established technology in downstream processing. The use of EBA has now proven successful in several applications at industrial scale and offers improved process economy and ease of operation. The first international conference on EBA was held in Cambridge, UK, December 1996 and the second in Napa Valley, CA, USA, June The third meeting in Germany has an expanded programme from Sunday through Wednesday. This will include experimental work, theoretical solutions, case studies from large scale industrial situations, results from different expression systems and further guidelines for the future development and establishment of EBA in biopharmaceutical recovery. Scientific programme The programme will include invited and submitted lectures as well as poster presentations covering the following topics: Theory of EBA (bed stability and expansion characteristics, adsorption performance) Case studies Scale up of EBA Regeneration procedures Validation and regulatory issues For further details write to: Conference Contact 42 Devonshire Road Cambridge CB1 2BL UNITED KINGDOM Announcement Or visit our web site. It will be regularly updated with information.

8 8 thirty It is now forty years since Swedish scientists Jerker Porath and Per Flodin introduced the pioneering technique of gel filtration with Sephadex. This was a significant development which laid the foundation for many of today's chromatography media. This article looks back at their work and the implications it has had on product development. The 40 th anniversary of Sephadex By Professor Jan-Christer Janson, Biomedical Centre, Uppsala University, Sweden Per Flodin in his laboratory. "We wish to report a simple and rapid method for the fractionation of water soluble substances" This quote is the opening sentence of the now classical paper by Jerker Porath and Per Flodin [1] who, in 1959, introduced gel filtration as a practical separation technique. In retrospect it must be regarded as one of the most significant advances made in the methodology of biochemistry. It had an enormous impact, not only scientifically, where the speed and simplicity of the new method contrasted strongly with most of the separation techniques then available, but also commercially. For Pharmacia AB (then a small pharmaceutical company in Uppsala, Sweden) the introduction of the first gel filtration medium, Sephadex TM, meant a pioneering effort that laid the foundation for the development of a large number of original products which are today invaluable to bioscience and biotechnology. So far, around 50 products (excluding those intended for use in health care and diagnostics) have been developed based on the epichlorohydrin cross-linked dextran gel Sephadex. Electrophoresis introduced The introduction of the gel filtration technique makes an extraordinary example of how scientific tradition and close links between academia and industry are all utilized in an optimal way. During the 1920s and 1930s, Professor The Svedberg and his pupil Arne Tiselius (Department of Physical Chemistry, University of Uppsala), introduced the techniques of ultracentrifugation and electrophoresis. They demonstrated how these techniques could be used for the analysis and characterization of biological macromolecules, revealing the complexity of natural protein mixtures, such as whole serum. Around 1940 Tiselius turned his interest toward chromatography. Systematic studies of adsorption chromatography led to the development of the elution, frontal and displacement methods. Tiselius also introduced the gradient elution technique. After having been awarded the Nobel Prize for Chemistry in 1948 for his work on electrophoresis and chromatography, Tiselius returned to electrophoresis; this time to explore the potential of zone electrophoresis. The technique was to be improved by his pupils Per Flodin and Jerker Porath who joined Tiselius in 1950 and 1952, respectively, and who worked together using starch and later cellulose as convection suppressors. Suggestion by Flodin There were many early accounts of sieving effects in the gel grains of the starch columns, but Porath and Flodin did not pursue any systematic studies of these at this time. In 1954 Flodin left Tiselius' laboratory to take up a position at Pharmacia; Porath continued with the development of column zone electrophoresis in vertical columns, now with cellulose as the main packing material. On the suggestion of Flodin, he also tried a powder obtained by grinding a block Reference [1] J. Porath, P. Flodin, Nature 183,1657 (1959).

9 thirty polymerizate of dextran obtained by cross-linking with epichlorohydrin. Flodin had synthesized this product after suggestion by Björn Ingelman, another pupil of Tiselius who had been employed as Research Director at Pharmacia AB in Dextran particles better Soon after, Porath could report to Flodin that he had observed the same sieving properties in the columns packed with the particles of cross-linked dextran as they had experienced some years earlier in the starch columns. However, the cross-linked dextran particles had much better physical properties than those of starch, and their porosity could be controlled with accuracy. The two scientists immediately realized that their discovery had important implications, both scientifically and commercially. Preliminary investigations gave such promising results that Pharmacia decided to file two patent applications. One for the gel products (March 1958) with Flodin and Ingelman as inventors, and one for the gel filtration method (April 1958) with Flodin and Porath as inventors. Soon after, the Pharmacia management decided to start product development. Birth of Sephadex The first two products were launched in the USA in July 1959 and were called Sephadex G-25 and Sephadex G-50 respectively. The name Sephadex came from SEparation, PHarmacia, DEXtran, G was short for Gel. The numbers stand for the water regain of the gels multiplied by 10. (Water regain is the maximum amount of water (in grams) taken up by one gram of dry xerogel). The separation ranges for these two gels are M r (G-25) and M r (G-50) for peptides and globular proteins, and M r (G-25) and M r (G-50) for polysaccharides. In 1960 the product range was extended to the less tightly cross-linked Sephadex G-75 with a separation range of M r for peptides and globular proteins, and for polysaccharides. At the time, all three Sephadex types were available in three different grades: Coarse ( mesh), Medium ( mesh) and Fine (< 200 mesh). These early products were all powders obtained by grinding dried, blockpolymerized cross-linked dextran. The particles obtained were irregular and easily generated fines, requiring decanting before column packing. The block-polymerization technique also proved to be inadequate for the production of Sephadex with higher G-numbers, even Sephadex G-75 was often problematic. 9 Fractionation range extended Electron micrograph of a dry Sephadex bead. Early product information for Sephadex in columns. In 1959 the development of bead polymerization procedures were initiated and in 1962 bead-polymerized Sephadex G-100 and Sephadex G-200 were introduced. With these two products, the fractionation range was extended to high molecular weight proteins and for the first time it was possible to fractionate complex protein mixtures, such as blood serum, into their main molecular size classes. By 1963, all five Sephadex types then available were produced by the beadpolymerization technique. Two years later, in 1965, the final Sephadex types were introduced: Sephadex G-150 (primarily intended for certain serum protein fractionations), the more highly cross-linked Sephadex G-10, and Sephadex G-15 for the separation of low molecular weight, water-soluble compounds. In the same year, all types except for G-10 and G-15, were made available in a superfine grade (10 40 µm). During the 1960s and 1970s thousands of references were published using Sephadex media. Sephadex was used in all kinds of epoch-making discoveries, including several connected with the Nobel Prize, as well as for the production of highly purified insulin for the treatment of diabetes. The year 1978 saw the launch of Sephadex IEF, a special grade of Sephadex G-75 for preparative isoelectric focusing, and in 1982 Sephadex G-50 Superfine Special Grade was introduced especially for industrial insulin purification. In 1985, for molecular biology applications, special DNA grades were introduced for the Sephadex types G-25, G-50 and G-100.

10 10 Today Sephadex media are also available in convenient prepacked columns. These include PD-10 Desalting, HiTrap TM Desalting, Fast Desalting and our most recent, HiPrep TM 26/10 Desalting, see article on page 28. All are packed with Sephadex G-25 and are ideal for sample clean up of volumes from 0.05 ml to 15 ml. A complete list of products based on Sephadex is given in Table I. Foundation for modern media Knowledge gained from Sephadex was also the basis of other modern media. Several other products are based on composite polymer media containing dextran. Sephacryl TM media were the first of these, launched in Sephacryl media are copolymerizates of N,N'-methylene-bis-acrylamide and allyl dextran. Superdex TM gel filtration media, composites of dextran and agarose were introduced in Ten years later came Sepharose TM XL and STREAMLINE TM XL, high capacity ion exchangers also based on composites of dextran and agarose. There are many factors that have contributed to the successful development of the market for Sephadex products. First of all, the new gel materials satisfied the demand for better tools for the purification of biological macromolecules. Their introduction was very timely too, at the very start of the rapid development of biochemistry and molecular biology in the early 1960s when many enzymes were being discovered and studied (mrna was discovered in 1961). Secondly, the technique was very simple and straightforward. Results were easy to predict and were very reproducible. The recoveries of biological activity were, with few exceptions, quantitative. Thirdly, the products could be developed, manufactured and marketed under a strong patent protection. Quality maintained The quality criteria for Sephadex products were set and the batch controls performed by people trained in a pharmaceutical company. The raw materials were quality controlled in the same way. This led to a reputation for consistent, high quality, from both a purity and a functionality point of view, which is still maintained today after 40 years on the market. Sephadex has resulted in the development of a complete range of products for purification of all types of molecules at all scales. Products which satisfy the stringent demands of the pharmaceutical industry and which are today important tools for the advance of science. Table I. Launch dates for Sephadex and Sephadex-based products Year Product Grades Block 1959 Sephadex G-25 Coarse; medium; fine Sephadex G-50 Coarse; medium; fine 1960 Sephadex G-75 Coarse; medium; fine DEAE-Sephadex A-25 Coarse; medium; fine DEAE-Sephadex A-50 Coarse; medium; fine 1962 CM-Sephadex C-25 Coarse; medium; fine CM-Sephadex C-50 Coarse; medium; fine SE-Sephadex C-25 Coarse; medium; fine SE-Sephadex C-50 Coarse; medium; fine Beads 1962 Sephadex G µm Sephadex G µm 1963 Sephadex G-25 Coarse ( µm); fine (20 80 µm) Sephadex G-50 Coarse ( µm); fine (20 80 µm) Sephadex G µm 1965 DEAE-Sephadex A µm DEAE-Sephadex A µm CM-Sephadex C µm CM-Sephadex C µm SE-Sephadex C µm SE-Sephadex C µm Sephadex G µm Sephadex G µm Sephadex G-25 Superfine (< 40 µm) Sephadex G-50 Superfine (< 40 µm) Sephadex G-75 Superfine (< 40 µm) Sephadex G-100 Superfine (< 40 µm) Sephadex G µm; Superfine (< 40 µm) Sephadex G-200 Superfine (< 40 µm) 1966 Sephadex LH µm 1967 Sephadex G-25 Medium ( µm) Sephadex G-50 Medium ( µm) 1968 QAE-Sephadex A µm QAE-Sephadex A µm 1970 SP-Sephadex C µm SP-Sephadex C µm 1975 BD-Sephadex µm Tracer Sephadex µm Tracer Sephadex µm 1976 Sephadex LH Sephasorb TM HP Ultrafine (10 23 µm) Sephadex IEF Cytodex Density Marker Beads Ten varieties 1981 Cytodex 2 Cytodex Sephadex G-50 Superfine special grade 1985 Sephadex G-25 DNA grade Sephadex G-50 DNA grade Sephadex G-100 DNA grade Prepacked columns 1970 PD-10 Desalting 1990 Fast Desalting PC 3.2/ HiTrap Desalting 1997 HiPrep 26/10 Desalting thirty

11 thirty 11 Intravenous Immune Globulin G (IGIV) is used to treat a broad spectrum of immunodeficiency syndromes including HIV indications, chronic fatigue syndrome, idiopathic thrombocytopenic purpura, as well as in bone marrow transplantation. Demand for IGIV has escalated over the past 20 years and currently exceeds availability. New production scale process for human IgG In an attempt to redress the imbalance between supply and demand for IgG, Bayer Corporation, Clayton, NC, USA, a leading producer of IgG in the US, has been investigating means of improving product recovery. The company has recently developed a method based on chromatography which gave a product with the same high purity as its proprietary IVIG-SD, but with a 50% increase in yield. During development of the method, the company screened 42 different chromatographic media; best results were achieved with Sepharose TM Fast Flow ion exchangers. There were several considerations in developing this new method. Bayer's current proprietary process purifies IgG from human plasma intermediates (Cohn Fraction II & III pastes) and these were to be the starting materials for the new method. It was also important for the process to integrate virus inactivation into the purification scheme. And finally, the new process had to be able to process large quantities, more than 200 kg paste per batch. Meeting the considerations Virus removal and inactivation are incorporated into the methods for dissolving the pastes. By adding the sodium salt of caprylate to the IgG suspension, lipoproteins are removed and enveloped viruses are inactivated. Precipitates are removed by filtration. The IgG-containing solution is at a low ionic strength, which is beneficial for chromatography since no additional treatment is necessary. Compatibility with the caprylate conditions was thus a condition for screened media. The chromatography step was designed for IgG flow-through and capture of impurities. Capturing IgG would require a high-salt elution buffer to elute the protein. The combination of high salt and high IgG concentration is believed to promote aggregation of IgG, which can lead to anti-complimentary activity and subsequent adverse reactions in patients when administered intravenously. Media screening was performed on columns with an internal diameter of 11 mm using standardised conditions for bed heights, ph, flow rates, conductivity and loading volumes. The different media were compared on the basis of their resulting product purity and yield. With the media selection procedure completed, the combination of column and medium was optimized. After the process was defined at lab-scale, the process was scaled-up in four steps for the production of clinical supply lots. Sepharose Fast Flow selected The chromatographic scheme comprised anion exchange followed by cation exchange: The first column contained a Sepharose TM Fast Flow strong anion exchanger, which bound all detectable albumin, most of the IgA and some IgM, as well as the majority of the caprylate. The second column contained a Sepharose Fast Flow weak anion exchanger and bound the remaining IgM and the rest of the contaminants. In addition, Bayer linked the two columns in series, which meant they could use smaller columns. Following chromatography the IgG solution is diafiltered, concentrated, and compounded for sterile dispensing. Significant improvements Bayer reports that the development of this chromatographybased method represents significant improvement over alcohol fractionation methods. Product purity and yield are high, neither a salt elution step nor a special step to remove the caprylate is needed, and column size is reduced. The method works with other combinations of strong and weak anion exchangers. The Sepharose Fast Flow resins were chosen because they had the best combination of performance and reproducibility. This is a summary of a presentation at Plasma Products Biotechnology '99, Daydream Island, Queensland, Australia March 1999: "Development and scale up of a production-scale chromatographic process for the pproduction of human IgG" Lebing, W.R., Lee D.C, Davies, A.O., Nixon, C.E., Paul, H.I. Bayer Corporation, USA. Reproduced with kind permission of the authors.

12 12 thirty Fast Trak Courses Fast Trak Courses are just one way Amersham Pharmacia Biotech shares its long experience of downstream processing. The courses we offer are designed to give researchers, development and production staff, as well as management, theoretical knowledge and practical experience in downstream purification processes. Practical courses cover column packing, basic chromatography, optimization and scale up. Theoretical courses cover chromatography and validation issues. Course schedule spring 2000 New this year in Europe Europe North America System Control using UNICORN in an industrial environment. SYS 1 and SYS 2. This course on UNICORN TM control software is designed to help you ensure that your ÄKTA TM, BioProcess TM, or other UNICORN controlled system is performing optimally. The course is at two levels, depending on your experience of using UNICORN and your needs (see opposite), and is run at the European Fast Trak TM Centre in Freiburg, Germany. Customized modules can be arranged at your premises. DEV 1 Mar 2-30 May Freiburg, DE San Francisco DEV 2 May 8-12 June Freiburg, DE San Francisco DEV 4 Feb Not applicable Freiburg, DE COL 1 Mar To be scheduled Freiburg, DE New!!! SYS 1 May Not applicable SYS 2 May Freiburg, DE VAL 1 Apr 5-6 To be scheduled Freiburg, DE Fast Trak Centres Fast Trak Purification services, including education, consulting, validation and regulatory support are available from Amersham Pharmacia Biotech throughout the world via the Fast Trak Centres. Contact the centres below or your nearest Amersham Pharmacia Biotech office. Europe Fast Trak Course Centre, Europe Amersham Pharmacia Biotech Europe GmbH Munzinger Strasse 9 D Freiburg Germany Tel: +49 (0) Fax: +49 (0) North America Fast Trak Course Center, USA Amersham Pharmacia Biotech Inc. 800 Centennial Avenue P.O. Box 1327 Piscataway, NJ USA. Tel: Fax: Japan Amersham Pharmacia BiotechK.K Sanken Bldg, 25-1 Hyakunincho 3-chome Shinjuku-ku Tokyo Japan Tel: +81 (0) Fax: +81 (0)

13 thirty 13 Course outlines Standard courses SYS 1 A two-day course for those who have some basic knowledge and experience of controlling a system with UNICORN, but who need further training to qualify for lab assistant, process operator or process supervisor. Subjects to be covered in theory and practice include: direct system control, method editing, creating methods and reports. GLP and current GMP issues relevant to working with a computer-related system form part of the course. Course code number: SYS 2 A two-day advanced course for those who have considerable experience (have a level equivalent to SYS1), but need a deeper understanding to qualify for development staff or system administrator. Subjects covered in theory and practice include: method writing and editing, use of conditional 'calls' and 'watch' instructions, administration, advanced evaluation procedures and reporting. System settings, network considerations and validation issues also form part of the course. Customized courses Customized training courses can deal with specific practical and theoretical topics within the field of chromatography. This is an excellent way to increase the efficiency of your team and to train personnel in order to comply with ISO 9000 and GMP requirements. Courses can be given at an Amersham Pharmacia Biotech training centre or at your facilities, depending on the equipment involved. New customized SYS modules In addition to the new standard SYS courses there are two customized modules intended to be run at customer premises. SYS B This is a short specialized course on "tuning" a BioProcess System, in other words, optimizing system variables and defining system settings. The course consists of lectures and practical demonstrations. Course code number: SYS C This is short, specialized lecture and workshop on issues related to using UNICORN in a network. Topics covered include UNICORN and validation, using UNICORN in an 0S/2 and in a Windows TM NT environment. Course code number: Please contact your closest training centre or Amersham Pharmacia Biotech office to discuss your requirements. Course code number: VAL 1 Validation Issues in Chromatographic Downstream Processing. A two-day seminar addressing key issues in validation of chromatographic units. Course code number: DEV 1 Process Development: Basic Practical Course on Chromatographic Methods. Three-day hands-on course gives an overview of chromatographic techniques. Course code number: DEV 2 Process Development: Practical Course on Chromatographic Methods. Four-day hands-on course in the design and optimization of a chromatographic process. Course code number: DEV 4 Process Development: Practical Course on Optimization and Scaling up to Full-Scale Production. Three-day hands-on course. Course code number: COL 1 Packing, Testing and Maintenance of Production Scale Columns and Standard Operation Procedures. Three-day hands-on course. Course code number: For information, please request the course announcement.

14 14 thirty Recovery of Biological Products IX Meeting report Whistler, Canada May 23-28, 1999 With around 40 oral presentations, 80 posters and a number of workshops it is impossible to do justice to all the contributors in one short article. This is a very brief overview of a stimulating and enjoyable meeting. Stuart Walker (Centre for Medicines Research, UK) set the scene with a look into the future of global drug development. Despite the fact that R&D funding rose from $20 billion in 1990 to $40 billion in 1998 there has been a slow but significant decline in NMEs (New Molecular Entities) over the past decade. Positive signs are an increasing success rate for clinical trials and the promise of genomics. The main challenges for the future are to maintain profitability, to discover innovative medicines, to carry out rapid drug development, and to harmonize the regulatory situation. Carsten Jacobsen (Novo Nordisk, Denmark) and Sean Dalziel (The University of Queensland, Australia) described crystallization processes for the recovery and purification of proteins. Crystallization was also the topic of a workshop. John Pedersen (Unizyme) presented a scalable process for production of human TNFa (Tumour Necrosis Factor) using a Histag fused to the target protein enabling purification in a packed bed on Chelating Sepharose TM Fast Flow or in expanded bed on STREAMLINE TM Chelating. Takao Hayakawa (National Institute for Health Sciences, Japan) discussed important components of the International Conference on Harmonisation (ICH) viral safety document and Arindam Bose (Pfizer) detailed the testing rationale. Joachim Walter (Boehringer Ingelheim Pharma, Germany) showed how microwave technology could be used for effective virus inactivation with retained protein stability. Affinity chromatography was a popular topic: Chris Lowe (Institute of Biotechnology, Cambridge), Inger Mollerup (Novo Nordisk), Ruben Carbonell (North Carolina State University) and Brian Kelley (Genetics Institute) all described novel affinity ligands for the purification of biopharmaceuticals. Alternative purification methods discussed included cross flow filtration, Ralf Kuriyell (Millipore), and aqueous two-phase partition systems, Andrew Lyddiat (University of Birmingham, UK). Andrew Russell (Eli Lilly) discussed strategies to accelerate the selection, development and commercialization of new biotechnology products. Richard Blackmore (Baxter) presented a small scale model of a pilot scale purification for recombinant haemoglobin automated using ÄKTA TM explorer 100. Aria Tavana (Biogen) described an interactive cost-modelling tool to allow process developers to evaluate the economic impact of different process alternatives. Karen de Jongh (Zymogenetics) discussed factors and trade-offs to be considered when selecting expression systems for novel recombinant proteins. Joanne Beck (Amgen) showed that cost savings of 20 to 25% resulted from using STREAMLINE TM for capture of CHO (Chinese Hamster Ovary) cell culture supernatant. Brian Turner (BASF Bioresearch Corporation) described the purification and characterization of anti-tnf from transgenic milk. The use of expanded bed adsorption for the capture of proteins from milk was also detailed in posters from Wim Noppe (Catholic University, Kortrijk) and Mark Etzel (University of Wisconsin. Lars Pampel (University College, London) demonstrated scale-down techniques to facilitate transgenic milk process design and Vikram Paradkar (Monsanto) gave a talk on the production and purification of proteins from transgenic plants. The workshop sessions (which unfortunately ran concurrently) included such wide ranging topics as outsourcing and technology transfer, chromatographic modelling, new chromatographic media, and process economics. For further information see the R9 Web site on Such was the interest that people were seen running from one session to the other, not wanting to miss anything. All in all a fitting tribute to R9. Even the sun shined!

15 thirty 15 Contract manufacturing - a way to get things done Speed to market is a key driving force in most industries: delays can spell disaster. The biopharmaceutical industry is no exception. The expansion in biotechnology has lead to numerous biological entities being available as possible drug development candidates. To avoid potential bottlenecks many companies are turning to outsourcing vendors or contract manufacturers for strategic stages of product development and manufacture. Contract manufacturing can enable a company to clear many hurdles in the race to market: new products can be introduced faster and at lower cost, whilst resources are not tied up and costly investments are avoided. In addition, companies which have developed competencies and facilities in areas such as cell culture, fermentation or downstream processing can exploit their investment despite variations in product flow down their own pipeline by offering contract manufacturing services. The extent to which contract manufactures are used varies enormously. One company may use one or more contract manufacturers for almost the entire development and production process, from early development through to clinical trails and full-scale manufacture. Another may use a contract manufacturer for only a particular stage in development or a step in production. Over the next few issues of Downstream TM we intend to run a series of interview articles from a number of contract manufacturers in Europe and North America. We kick-off with a report from Covance Biotechnology Services, North Carolina, USA and Avecia, (formerly Zeneca LifeScience Molecules, Biologics) Cleveland, UK.

16 16 thirty As part of a series of articles highlighting contract manufacturing, Downstream talked to several members of the process development team at Covance Biotechnology Services. Bob Boucher, Philip Ropp, and Scot Shepard are part of a 17-member team which evaluates, optimizes and develops bioprocesses on behalf of client companies. The team is highly qualified and experienced and well able to meet the process development challenges offered by the variety of recombinant proteins in discovery today. A talk with Covance Biotechnology Services In general terms, what can Covance offer biotech companies planning to outsource biomanufacturing? "We transfer and scale up a client's bench scale bioprocess method to a validated process in our cgmp (current Good Manufacturing Practices) manufacturing area where production of clinical trial material is performed under rigorous control. Here we work at a scale ranging from very small bioreactors (10 litres) to large scale vessels of litres or larger. Our group's objective is to develop processes that provide the highest quality product at the lowest possible production cost whilst meeting the customer's requirements for delivery dates and timeframes. For our large scale applications we usually rely on BioProcess TM Systems, but the client ultimately makes the decision on the final process and equipment. We have several BioProcess Systems in the Phase I production and large scale manufacturing areas." At what scale do you develop bioprocesses? "The team has first-hand experience developing purification processes for a wide range of scales. We scale up using ÄKTA TM explorer, often starting with columns like HiTrap TM (1 ml and 5 ml) columns with flow rates from a fraction of millilitre, before continuing on to BioProcess systems and considerably higher flow rates. We use ÄKTAexplorer because it's flexible, fast and relatively inexpensive compared with the higher costs associated with running a process in the large scale cgmp facilities. We have 25 ÄKTAexplorer systems in the process development area." ÄKTAexplorer in the process development labs. Why is the speed and flexibility of ÄKTAexplorer useful for process development? "Speed to market is an important driver in pharmaceutical development and our clients expect us to deliver a high quality product (their product) in a relatively short time frame. Typically a client's process has to be evaluated, optimized and developed, (leading to a production batch being available for toxicity studies) within a few months. ÄKTAexplorer is compatible with these tight project timelines enabling the team to get results faster. Also, you're up and running quickly with ÄKTAexplorer. Its flexibility helps improve our operational efficiency. For example, a programming feature of ÄKTAexplorer enables the screening of a series of different purification columns overnight so that the analysis can be completed the next morning." Is there anything else you like about ÄKTAexplorer? Bob Boucher likes the range of flow rates available and he uses UNICORN TM software because he can use the same software from process development to production. Bob works mainly with evaluating purification methods using HiTrap, HR 5/5, HR 5/10 and HR 10/10 columns with a variety of

17 thirty media including ion exchange, hydrophobic interaction and affinity resins. The evaluation of purification media can take anywhere from 2 weeks to 2 months in order to identify the best resin and establish robust conditions for purification. It all depends on the complexity of the product. "It's important to have a cost-effective process which means having a minimal number of steps. ÄKTAexplorer has proved trouble-free and UNICORN is easy to learn and use. The integration of process development on ÄKTAexplorer with bioprocessing through UNICORN is useful. We spend the time up-front refining a purification process on ÄKTAexplorer because we need to have the protocol work in the manufacturing floor with BioProcess System. There's an advantage to be able to use the same software." Philip Ropp mentioned that scientists with limited experience are able to run UNICORN, using the built-in methods provided with the system. "It's fairly easy for beginners to be up and running on UNICORN." Philip also appreciates the flexibility of applying ÄKTA to a variety of purification tasks. For example, he uses adsorption at 260 nm and 280 nm to monitor the removal of contaminating DNA from the protein product. He also saves all his experimental data forming a reference database. Every project brings new challenges as unique as the proteins themselves. Scot Shepard described how Covance used STREAMLINE TM expanded bed adsorption to resolve problems purifying large scale batches of recombinant proteins. The protein of interest is expressed and secreted into the growth medium requiring it to be separated from host cells. Can you describe the type of problem you have overcome in development? "The process, developed by the client, used bench-scale centrifugation followed by a number of filtration and chromatography steps. The recovery process would have taken 36 hours to perform at scale, which was too long considering the protease content of the crude feed. A major challenge was figuring out a way to work with the high percentage of solids following fermentation, which can be 45% of the total volume. This challenge was compounded by a need to ultimately provide large scale purification. From our experience, we know that as a client's product progresses through clinical trials, there is going to be a demand for larger batch quantities. This meant that we were going to be running at a litre scale of fermentation or larger, which would be rather costly. Fortunately, we were able to modify the process such that recovery, clarification and initial purification of the product was accomplished using STREAMLINE SP. We used conventional chromatography steps for final purification. The new process now takes 8 to 12 hours to perform and the recovery efficiency is quite high compared with the previous method." Can you tell us more about recovery? "Recoveries now average greater than 95%, which is much higher than the results using centrifugation. This is because a significant amount of supernatant was trapped in the centrifuged cell paste and was lost going into the purification step using the old method. Using an expanded bed method avoids this loss." At what scales are you working? "The client transferred a 10 litre fermentation process which we initially developed to run at a 140 litre fermentation scale using a STREAMLINE 200 column. We developed a linear scale up with a bed height of 20 to 25 cm. For large scale production, we re using litre fermentation runs and a STREAMLINE 600 column. The total volume of sample applied to the STREAMLINE column is about litres, which is reduced to about 150 litres following the STREAMLINE step. The flow rate is 15 l/min. The product is stable and greater than 80% pure after the expanded bed chromatography step." Can you describe your CIP procedures "We developed a novel cleaning method for the adsorbent and hardware which works for more than 25 cycles at the litre scale." And what about the future? "We have experienced success applying STREAMLINE to this reproducible process and a second STREAMLINE process has recently been transferred to manufacturing at the litre scale. What is interesting is that we are finding that clients are also using this technology in the processes they employ involving biopharmaceuticals." Covance Biotechnology Services, located in Research Triangle Park, North Carolina, USA, is a part of Covance Inc. As a contract manufacturer of biopharmaceuticals with cgmp manufacturing, Covance offers clients state-of-the-art facilities and expertise dedicated to the production of biotechnology products using mammalian cell culture and microbial fermentation. Process development services focus on optimization of gene expression during cell culture, downstream protein processing, purification, protein characterization and product formulations. The company can produce a wide range of therapeutic products including monoclonal antibodies, growth factors, cytokines, recombinant vaccines and nucleic acids for clinical trials and commercial markets. 17

18 18 thirty Re-designed process Avecia (formerly Zeneca LifeScience Molecules Biologics) was approached by a client for help to improve the productivity of a process for Phase II & III clinical trials. Below, Avecia describes how it addressed several problems and re-designed the process. The improvements gave a 12-fold increase in productivity. A vecia has recently completed a project for a client which resulted in a 12-fold increase in productivity for the production of a molecular weight protein. The client approached Avecia with a process that had been used to produce material for Phase I trials, but which did not give the required purity for Phase II and III studies. Furthermore, recovery was extremely low, approximately 25%, and it was virtually impossible to scale up the process. Thus, in order to carry out Phase II and III studies, the process needed improvements addressing robustness, scalability and purity. In addition, the current process was rather lengthy, requiring up to 10 days per batch. Areas to address FineLINE 200 and BioProcess system were used in the pilot scale process. Introduced STREAMLINE The fermenter for the pilot plant process. The goal was to design a scalable process, which could provide the quantity and quality (higher purity) of product for Phase II and III trials. Three areas required to be addressed: improve the analytical method and develop a validation package for the assay improve, optimize and scale up the fermentation process develop a downstream process which was scalable, robust and would deliver the required purity (>98%) Dr John Liddell, Head of Separation Science, described the project. One of the first things we did was to carry out a technology transfer of the old process and analytical methods. This indicated that a 8 to 10% improvement in purity was required over the old process. Secondly, we looked at the fermentation process, which yielded a low titre. By developing an understanding of the key fermentation variables followed by optimization, we were able to increase the fermentation titre by a factor of three. The next step was to examine the existing downstream process, which was based on traditional chromatography methods and technologies. We came up with two major changes, one in the capture step and one in the intermediate purification step." In the original process, fermentation was followed by two membrane filtration steps, and then packed bed ion exchange chromatography, see Figure 1. The overall recovery over these steps was 70%. "We believed that STREAMLINE TM expanded bed adsorption was a better alternative. Only 4 to 6 weeks of experimental work enabled us to verify that STREAMLINE was indeed an excellent choice. Its introduction into the scheme not only increased recovery to 95%, but also reduced the number of steps from three to one, replacing the two filtration steps and the packed bed ion exchange step," said Eleanor Clements, Senior Development Scientist. (See Table 1.) Table 1. Improvements made in the individual chromatographic steps. Individual steps Original New process process Capture step STREAMLINE Recovery 70% 95% No. of steps 3 1 Intermediate purification step Silica-RPC SOURCE 15PHE Number of runs/batch 6 1 Recovery 40% 85% Purity 85% 95% Loading capacity 3 g/l medium 12 g/l

19 thirty increased productivity 12-fold 19 ORIGINAL process Fermenter NEW process Fermenter Capture Micro filtration UF/DF Anion IEX Capture Intermediate purification STREAMLINE SOURCE 15PHE Intermediate purification Polishing Cation IEX Silica-RPC Cation IEX Polishing UF/DF SOURCE IEX UF/DF UF/DF Figure 1. Flow schemes of the original and new processes. The intermediate purification step used reversed phase chromatography on a silica matrix. "Initially we tried to see if we could improve purity by altering loadings, flow rates, and by changing solvents. The results were disappointing. The required purity could not be achieved and loading capacity remained low, 3 grams per litre of medium. The loading capacity of the RPC medium was a real stumbling block. Despite the size of the column in use at pilot scale (2 litre bed volume and i.d. 110 mm), the RPC step needed to be performed six times at the 100 litre fermentation scale. Imagine the size of column needed and the number of cycles per batch if you decided to use a 1000 litre fermentation." commented John. "Another concern was the poor recovery of protein from the column, which is typical for silica-based RPC matrices," he added. HIC gives required purity How could the required purity be achieved? "We were reluctant to replace the RPC technique at this point as it was the only method known to give significant purification of the product. Consequently, we tested other RPC media, but without success in meeting the purity target. We then opted for hydrophobic interaction chromatography as it is based on the same principles of separation as RPC, but conditions are much milder and no organic solvents are necessary. Colleagues of ours in our DNA medicines group had successfully purified oligonucleotides on a high resolution ion exchange medium, SOURCE TM 15Q. So we decided to test the high resolution SOURCE HIC media," John explained. "We screened the three HIC media, SOURCE 15ETH, SOURCE 15PHE and SOURCE 15ISO, under a range of solution conditions which identified SOURCE 15PHE as being the most suitable. Comparing runs on SOURCE 15PHE with runs on the silica RPC medium gave some very interesting observations: loading levels on the HIC medium were four times higher than on the silica-rpc medium. In addition, SOURCE media proved to be easy to scale up. The high loading capacity and larger SOURCE 15 PHE column allowed each batch to be handled in one run rather than 6 runs of the original process. It took us only 14 days to optimize the chromatographic conditions for operating the SOURCE 15PHE purification step," Eleanor added.

20 20 SOURCE gave immediate improvements "After scaling up to pilot scale, the difference in results between the silica-rpc and the HIC steps were dramatic: using silica-rpc with solvents acceptable for large scale operation, you'll recover at best 40% of the material with a purity of 85%. In contrast, using SOURCE 15PHE you'll recover 85% of the material with 95% purity," remarked John. (This is illustrated in Figure 2, which shows the purity and yield in fractions from pilot scale runs using silica RPC media and SOURCE 15PHE.) He went on to say, "SOURCE media proved to be very easy to scale up. We have data showing that the elution profiles at different scales are virtually identical. The shapes of the peaks are very similar and the conductivity gradient is consistent and reproducible. The media also proved to be very easy to pack into the FineLINE TM columns used - a big advantage over packing silica RPC media which requires the use of solvents such as pure isopropanol or pure ethanol." "Another benefit is that when using SOURCE 15PHE it is possible to use the same NaOH sanitization method for all steps in the process, which facilitates validation. This is not the case if a silica RPC medium is used," remarked Eleanor. thirty The introduction of STREAMLINE for capture and SOURCE 15PHE for intermediate purification made the whole process much simpler, more cost-effective, and easier to move the product forward on to the next stages. The re-designed process is now scalable up to a 1000 litre fermentation scale, and the processing time has been decreased from 10 to 5 days. The overall productivity has increased 12-fold, purity has improved and the overall process economics are better, see Table 2. Table 2. The re-designed process. Productivity has been increased 12-fold. Overall process Original process New process Improvements Number of steps 7 5 Fermentation 3-fold titre increase Recovery 25% 45-50% 2-fold Time 10 days 5 days 2-fold Productivity 12-fold increase Purity 90% 98% +8% Reproducible "The process is now more compact and can be run on standard equipment that does not require high pressure pumps and use of solvents. The process is very reproducible," said Eleanor. Yield (%) RP-HPLC SOURCE 15PHE "This experience will really make us consider SOURCE and STREAMLINE a first choice for future applications. This particular process has truly demonstrated that high resolution separations are achievable with SOURCE media," concluded John Purity (%) Figure 2. Comparison of yield and purity fractions from HIC and RPC steps. The columns used for HIC had a diameter of 200 mm compared with the original RPC column of 110 mm. Avecia (formerly Zeneca LifeScience Molecules Biologics) was formed in the mid-nineties to meet process development and manufacturing needs of the pharmaceutical, biotechnology and agrochemical industries. It employs over 500, split over several sites. As a contract manufacturer it offers services in fermentation, characterization studies, process development, QC development and validation of equipment and processes. Achieved goals Avecia was very happy with the outcome of the project. It had achieved all the goals set by the client and within the project timeframes. The development of the revised process started in June 1998 following technology transfer activities. By February 1999 the pilot plant was operating the re-designed process which is now yielding the required purity and quantity of product necessary for Phase II and Phase III studies.

21 thirty 21 The Fourth Waterside Monoclonal Conference Process development and production issues for monoclonal antibodies Norfolk, Virginia, USA April 12-15, 1999 Meeting report For the Fourth Waterside Monoclonal Conference the majority of the lectures focused on upstream processing. Participants came from industry, academia and state-funded institutes. Cell culture Catherine Bentley (Glaxo Wellcome) and Jeremy Wayte (Lonza Biologics) stressed the importance of generic processes to ensure speedy supply of product for early phase clinicals and described the subsequent strategies necessary for process optimization (e.g. cell line improvement and culture medium development). Kathie Fritchman (PharMingen) and Bill Bucher (Lampire Biological Laboratories) had evaluated a novel cell culture medium for high yield production of MAbs using a variety of culture systems. Mina Mahadevan (Medarex) described serum-free media development for MAb production in high cell density perfusion bioreactors. Paul Sauer (Process Design Labs) Thomas Seewoester (BASF Bioresearch Corporation) and Wheichang Zou (Merck) all described the development and advantages of fed batch processes and Yeng-Dar Yang (Immunomedics) reported increased productivity using a fed perfusion process. Matthew Jerums (Amgen) described monoclonal antibody production using the Cytopilot TM Mini fluidized bed reactor together with Cytoline TM microcarriers. Process development Helen Chadd (Abgenix) presented the process development, scale up, manufacture and characterization of two human monoclonal antibody products using XenoMouse TM technology. Eric Tsao (MedImmune) discussed the key issues involved in advancing from early feasibility stage to large scale commercial manufacturing (cell culture, purification, viral clearance) at multiple sites. Richard Francis (Glaxo Wellcome) stressed the importance of understanding how process conditions impact on product quality in the manufacture of MAbs. It is vital that this information is obtained during process development. Gerardo Zepata (Genentech) described the successful recovery of recombinant antibodies from unclarified Chinese Hamster Ovary Cell Culture Fluid using STREAMLINE TM Protein A. Gerry Bell (Boehringer Ingelheim) presented a non-affinity approach for purification of monoclonal antibodies. Dennis Michiel (SAIC-Frederick, NCI-FCRDC) described the purification of clinical grade murine IgG 1 including a generic template for murine IgG 1 purification development. Martin Vanderlaan (Genetech) presented a generic approach to Host Cell Protein impurity testing in antibody products. Having one assay applied across products allows comparisons between products and processes and facilitates development of new products and improvement in existing products. He demonstrated clearance of representative CHOPS (Chinese Hamster Ovary Cell Proteins) over 5 to 6 logs. Regulatory issues Marjorie Winkler (Genentech) discussed the process validation programme for the Lenercept recovery process. Paul Dal Monte (SmithKline Beecham) described formulation development studies conducted in support of a dosage form for a therapeutic monoclonal antibody in clinical development. Kathleen Grant (BASF) described the characterization of recombinant human monoclonal antibodies. Kurt A. Brorson (FDA/CBER) gave a talk on the current status of CBER policy on comparability protocols for manufacturing changes. The conference was well attended with about 200 participants coming mainly from North America.

22 22 Intensification of a large scale thirty The growing demands for MAbs has put pressure on manufacturers to increase production. Here, Centocor BV, The Netherlands, describes how new technologies introduced into an existing process increased yield by 25% and decreased process lead times by 50%. The improvements also enabled Centocor to utilize the capacity of the manufacturing plant better. The novel packing technology used in CHROMAFLOW facilitated the use of large diameter columns. This article is based upon material from Dr Richard Francis, formerly Senior Manager, Operations Technical Support at Centocor, The Netherlands. Dr Francis is now at Glaxo Wellcome, UK. Monoclonal antibodies (MAbs) came of age as therapeutic products in the early nineties. In the past decade the requirements for purified MAb have advanced exponentially in order to meet the ever growing clinical and commercial demands. Original MAb preparations were the result of in vivo production, yielding milligram to gram quantities. Today large scale cell culture techniques using continuous perfusion fermentation can yield multiple kilograms MAb per bioreactor. The challenge is thus to develop and scale purification processes that are capable of yielding kilogram quantities of purified MAb at the lowest possible cost. The scale up programme performed at Centocor resulted in a very large increase in batch size. The successful re-engineering of the process resulted in a yield increase of 25% and a decrease in processing lead times of 50%", reported Dr Francis. "In addition, this approach also results in a better utilization of manufacturing plant capacity". Identifying key factors Dr Francis went on to describe how Centocor's experience with one MAb purification process resulted in the successful upscaling of a purification process from 250 grams to 8 to16 kg processed per purification batch. The key factors in facilitating this upscaling process and intensification program were: Elimination of process steps such as diafiltration (membrane ultrafiltration) with in-line product dilution using chromatography. Utilization of high binding capacity media (Q and SP Sepharose TM High Performance) to increase product loading from 20 to 100 grams MAb per litre of medium. Utilization of a new chromatography column design (CHROMAFLOW TM ), to ease operation considerations for the use of 1 metre diameter columns. This technology enables in-situ column packing/unpacking. Introduction of direct product recovery from the bioreactor harvest stream, seamless integration of the fermentation and purification processes. Expanded bed adsorption chromatography using STREAMLINE TM is being tested. This overall approach is best illustrated by a direct comparison of the original 250 gram process, with the intensified 8 to 16 kg process, Figure 1.

23 thirty commercial monoclonal antibody purification process 23 ORIGINAL 250 g purification process Bioreactor yielding approximately grams MAb Harvest concentration by ultrafiltration Protein A chemistry Virus inactivation step Primary ion exchange step, virus inactivation step Concentration and diafiltration by ultrafiltration then secondary ion exchange step Concentration and diafiltration by ultrafiltration then third ion exchange step Concentration and diafiltration by ultrafiltration then gel filtration chromatography Diafiltration and final product formulation INTENSIFIED 8-16 kg purification process Bioreactor yielding approximately grams MAb Direct product capture from bioreactor harvest using STREAMLINE rprotein A Virus removal filtration and inactivation treatment Primary ion exchange step Secondary ion exchange step In-line dilution third ion exchange step Ultrafiltration final product formulation Figure 1. Original 250 gram purification process compared with the intensified process. One bioreactor to one purification lot "The original process achieved 40% MAb recovery from the bioreactor harvest to final product formulation. Another important point from a manufacturing plant perspective is that > 32 x 250 gram batches are required to process the output from one bioreactor. The intensified purification process is scaled such that 8 to 16 kg product can be processed per purification lot. Overall yield from bioreactor harvest to final formulated product is 75%. Of importance is the fact that the fermentation and purification operations are more balanced, i.e. one bioreactor = one purification lot," Dr Francis pointed out. Characteristics unchanged During the scale up, batches are subject to testing to ensure that the product's characteristics are unchanged. Studies are performed to test product identity, purity and potency as well as evaluate removal of impurities and clearance of viruses. The results are compared with the previous scale of operation. In the current example, the direct product capture step using Protein A chromatography is still performed in the standard axial packed bed configuration. At present, the harvest is clarified using depth filtration, which represents a significant cost. The next step will be the introduction of an expanded bed methodology in an attempt to further reduce manufacturing overheads by eliminating the harvest clarification filters. Critical approaches Dr Francis summed up the experience, "As you can seen from this example, such process intensification/upscaling approaches are critical if we are to meet the ever increasing demands for purified Mabs. The correct application of such strategies can be a major factor determining the success or failure of a product in a commercial environment, and will certainly have an impact on the 'bottom line'". Centocor is a health care company specializing in the development and commercialization of therapeutic products to meet critical human health care needs. A leader in monoclonal antibody technology, Centocor's innovative products focus on the management of three major disease areas: cardiovascular, autoimmune and cancer.

24 24 thirty A recent trend in downstream processing is to use affinity chromatography as a first step in the purification scheme. This is advantageous since an effective capture step has a major impact on the subsequent purification, usually offering a very compact process with high product yield and unmatchable purity. In order to apply affinity chromatography to more processes, a system for developing novel ligands is required since naturally occurring ligands are sometimes difficult to isolate and often unstable. Collaborations to generate customized ligands At the 13 th International Symposium on Affinity Technology and Bio-Recognition, July 4-8, 1999, there was much discussion around "designer ligands". Of a number of approaches discussed and presented, two appear to be particularly attractive: combinatorial libraries and phage display. ArQule Inc. (Medford, Massachusetts, USA) is a young company with unique ability to synthesize and screen organic compounds. It has generated hundreds of thousands of novel, diverse, small organic compounds with potential affinity for biomolecules. The small ligands are suitable for early capture directly from the fermenter due to the highly stable organic molecules. Joe Hogan from ArQule together with Åke Pilotti from Amersham Pharmacia Biotech gave a presentation entitiled "Discovery of novel affinity chromatography ligands using combinatorial chemistry and rational design". They discussed the background to ligand development in drug discovery and its application in chromatography today. Sometimes a larger ligand molecule will be required to achieve the binding affinity and chromatographic selectivity required to solve a difficult purification problem. Another company, Dyax Corp., (Cambridge, Massachusetts, USA) offers proprietary phage display technology which allows the development and screening of peptide libraries containing millions of molecules which may fill this bill. At the same meeting, James Booth of Genetics Institute presented his work "Identification and development of a peptide affinity ligand for the purification of a recombinant therapeutic protein using phage display" (based on a collaboration between Genetics Institue and Dyax Corp). These kinds of customized peptide ligands show a very high degree of selectivity and in this presentation rfactor VIII was successfully isolated. The use of organic molecule ligands and phage display peptides can offer benefits of higher yields, greater purity, and fewer process steps. In order to make these elegant approaches viable in terms of robust media for industrial production, Amersham Pharmacia Biotech has made collaborative agreements with both ArQule Inc and Dyax Corp. Our intention is to be able to offer tailored and cost-effective solutions to difficult separation problems, either on an exclusive basis, or as regular products.

25 thirty 25 Press release Amersham Pharmacia Biotech and Dyax Corp agree to develop and market innovative separations technology Cambridge, MA, US, August 26, A commercial agreement between Amersham Pharmacia Biotech and Dyax Corp., of Cambridge, Massachusetts, US, will allow pharmaceutical and biotech companies to isolate biomolecules of interest more effectively a crucial part of the research, development and manufacture of drugs. Under the agreement Amersham Pharmacia Biotech and Dyax will jointly develop affinity products for the purification of selected biomolecules, offering both off-theshelf and customised solutions for biopharma companies. The agreement combines Amersham Pharmacia Biotech's market leading technologies for purification with Dyax's innovative Affinity by Design TM ligand discovery and development capabilities based on Phage display. The affinity media products resulting from the collaboration will help biopharma companies to solve previously intractable problems and to reduce the number of steps involved in separating biomolecules of interest be it for research or production processes - saving time in development and cost in production. "This is a major opportunity for Amersham Pharmacia Biotech, extending our ability to provide customers with tailored and more cost-effective separations solutions," said Ingvar Wibeger, vice president, Industrial Separations, at Amersham Pharmacia Biotech. "With post genomic research revealing new proteins and an increase in the number of biopharmaceuticals in clinical trials, pharmaceutical and biotech companies are under growing pressure to speed up purification and improve the efficiency of downstream production processes." An international symposium on downstream processing of genetically engineered antibodies (GAb's) and related molecules (GAb 2000) Announcement Barcelona, Spain October 15-17, 2000 Fifteen years have gone by since the Nobel Prize was awarded for discovery of the principle for generation of monoclonal antibodies. Today, "monoclonals" represent one third of all biotechnology drugs under development. Most of them are genetically engineered and include humanized antibodies, fusion proteins and fragments. This symposium will address critical issues related to the downstream processing of such molecules. Note this event in your agenda for year 2000 and watch the web site (www. conferencecontact.co.uk/gab2000) for your invitation to submit posters and oral papers. GAb 2000 will cover: Humanized antibodies, antibody fragments, Fc and Fab fusion products, and GAb conjugates New approaches to GAb purification Downstream modification of GAb's Structure and function of affinity ligands for GAb's The organizing committee is represented by: Dr Joachim Walter (chair), Dr Hermann Allgaier, Dr Steven Chamow, Ms Gail Sofer and Professor Mathias Uhlén.

26 26 thirty The 16 th ESACT meeting Products from cells, cells as products Lugano, Switzerland Meeting report April 25-29, 1999 Lugano, Switzerland was the setting for the 16 th meeting of European Society for Animal Cell Technology, ESACT. Well over 600 attended the four-day meeting, which was divided into nine sections and supported by 56 exhibitors. The first section was entitled: Cell physiology, genetics, and vectors for efficient product formation in animal cells. The emphasis of the presentations here was on increasing product yield and product quality. Various methods had been evaluated, including enhancers, inducible expression systems, ways of affecting the post transitional modification and metabolism of cells, novel suspension culture as well as continuous annular chromatography to increase product quantity/quality. The second section: Novel therapeutic and prophylactic approaches based on cells and nucleic acids, discussed therapeutic tools and included nonviral tools for gene delivery, lentiviral vectors, tissue engineering of a bladder patch, encapsulation of cells for type I diabetes treatment, allogenic bone marrow processing, use of amniotic epithelial cells in transplantation as well as large scale adenovirus production for gene therapy. The focus of presentations in the third section, New cell lines/technologies for stable and transient production of proteins, was mainly on production issues issues concerning baculovirus vectors, using p35 to increase viability of virally infected cells, transient expression, HEK suspension cultures, transgene expression control and the importance of high product expression. Cells and product of cells in biosensor development and nanotechnology was the topic of the fourth section which covered microfabrication, single molecule spectroscopy by AFM, Green Fluorescent Protein (GFP) producing cells for growth assessment, and orally absorbable antibiotics in Caco 2 tests. Section 5 was entitled: Vaccines and Immunologicals. Topics covered in this session included MAbs for human therapy, IgG 1 glycoforms for optimal cellular cytotoxicity, chimeric lyssavirus glycoproteins used in multivalent vaccines. Influenza production in MDCK cells in suspension, maximization of virus like particles (VLPs) in insect cells, recombinant a-rhd prevention of haemolytic disease in newborn were also discussed. Animal or patient derived cells and tissues for therapeutic applications was the title of the sixth section. Heamatopoetic stem cell expansion (CD34+), osteogenesis, myocardial tissue replacement, keratinocyte culture as well as cardiomyocytes were all tissues and cells that had been explored as potential sources for therapeutics. DNA sequencing, genome analysis and animal cells, Section 7, was devoted to a new alphaviral expression screening technology called DELphi. Section 8: Cell characterization, validation, safety, adventitious agents. The session took up the topical subject of status of the ICH harmonisation process, as well as regulatory standards for gene transfer protocols in Europe, risks with porcine xenotransplantation, biopharmaceuticals in transgenic milk, cryopreservation of protein-free media, and considerations for construction of a clinical phase I/II manufacturing facility. The final section covered Integration of biology and technology. Process development and downstream processing. This session discussed topics such as the optimal perfusion process, factors affecting glycosylation, sialidase impact on product quality, feedback control, mathematical models, air-trap technology, aeration of 8000 litre vessels, transient gene expression and retrovirus production. The next meeting of the European Society for Animal Cell Technology, ESACT, will be held in Tylösand, Sweden, 2001.

27 thirty 27 Towards harmonization of drug regulation Attempts are being made to harmonize some regulatory procedures in order to speed up assessment of new drug applications and accelerate new drug development. In 1990 a tripartite harmonization forum was established between Japan, the USA and the European Union. It is known as the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). This tripartite forum has worked together to produce technical guidelines regarding new drug development and regulatory requirements. The guidelines, 16 quality, 12 safety and 12 efficacy, aim to eliminate redundant and duplicative technical requirements in the participating countries with respect to the development and approval of new medicinal substances and products. The guidelines address not only the efficacy, safety and quality of pharmaceutical products, but also cover biotechnology products, regulatory terminology and electronic standards for transmission of regulatory information. All ICH countries are committed to implementing these guidelines, which means that there are now areas of drug regulation that have become harmonized in the countries involved. However, the guidelines are not comprehensive in covering the totality of the requirement for quality, safety and efficacy. Gaps in the guidelines must be filled by national regulations. Today, WHO is the only intergovernmental health organisation with a world-wide responsibility for harmonization of health-related issues. As such it acts as a bridge between the ICH and non-ich countries to disseminate information and to promote understanding of the applicability of ICH products. The ICH comprises 17 member countries, non-ich members total 176. The sheer number of countries involved gives an idea of the enormity of the task for global harmonization. Given the fact that not all regulatory requirements and procedures can be harmonised due, for instance, to different climatic influences, it is more likely that there will be regional harmonization, such as that going on in the European Union. Communication will take place between these regions enabling both developed and developing countries to participate in the process to reduce world-wide regulatory disharmony. Reference Innovations in Pharmaceutical Technology, Vol. 1, Issue 2, The bitter sweet life of Miraculin In Downstream 21, we ran the press release from BioResources International (BRI) Ghana, Ltd which described its breakthorough in extracting and purifying the taste-masking protein, Miraculin TM. This protein has the uncanny ability to make sours things taste sweet. Purified Miraculin is today in high demand as an ingredient to change the taste of bitter tasting foods, medicines, wines, confectionery, etc. In a technical partnership agreement with BRI, Amersham Pharmacia Biotech provided the technical expertise and support for the final purification and large scale processing of this protein. In March 1999, the process for the purification of Miraculin glycoprotein using tandem hydrophobic interaction chromatography was granted the following patent: US 5,886,155.

28 28 thirty HiPrep 26/10 Desalting is an excellent alternative to dialysis in the research lab, especially when larger sample volumes are used or when sample degradation may occur with time. By coupling four columns in series, 60 ml of sample can be processed in 20 to 30 minutes, with high flow rates and without encountering back-pressure problems. Figure 1. Four HiPrep 26/10 Desalting columns in series on ÄKTAexplorer. HiPrep 26/10 Desalting columns Buffer exchange and desalting are common operations in any laboratory engaged in sample purification and analysis. It is often necessary to change the buffer composition of a sample between chromatography steps or to satisfy special requirements of an assay. Desalting of a sample is a prerequisite for mass spectroscopy analysis, lyophilization, and after certain procedures such as ion exchange chromatography. Although they can be accomplished by dialysis, this is a timeconsuming process, and samples sensitive to degradation may be at risk. Because of its high speed and high volume capacity, HiPrep TM 26/10 Desalting is an excellent alternative to dialysis, especially when sample volumes of 15 ml are used or when samples need to be processed rapidly to avoid degradation. Samples of 15 ml (30% of the total bed volume) or less can be applied to a single column, and by coupling up to four columns in series, see Figure 1, a maximum sample volume of 60 ml can be used. Even with four columns in series, high flow rates can be maintained without back-pressure problems with equipment such as ÄKTA TM design system, resulting in fast separations. In fact, in 20 to30 minutes, up to 60 ml of sample can be desalted or buffer-exchanged. Each HiPrep 26/10 Desalting column is packed with Sephadex TM G-25 Fine for rapid, reproducible and easy separations. The cross-linked dextran beads that comprise this medium allow for excellent resolution and high flow rates. Globular proteins with molecular weights between and fall within the fractionation range of the matrix. Proteins/peptides with a molecular weight greater than can be separated from molecules, such as salts, whose molecular weights are less than Other characteristics of the HiPrep 26/10 Desalting column are listed in Table 1. Table 2 shows how sample volumes can be scaled up using HiPrep 26/10 Desalting columns in series. Samples in these tests consisted of either 30 or 60 ml of a fungal culture supernatant, containing a secreted recombinant protein. Samples were run on two (30 ml sample) or four (60 ml sample) columns connected in series. In addition to HiPrep 26/10 Desalting, Amersham Pharmacia Biotech offers a range of columns for desalting and buffer exchange that can accommodate variations in sample size and yield acceptable levels of dilution. Please contact your local Amersham Pharmacia Biotech office.

29 thirty 29 5 th INTERLAKEN CONFERENCE on Advances in Production of Recombinant Proteins Interlaken, Switzerland March 28-31, 2000 The 5 th Interlaken Conference will continue the traditions of scientific excellence, industrial experience and open communication which have become the hall marks of the conference series. The conference will focus on the topics of expression, downstream processing analysis and regulation. Early registration and submission of papers by 13 December For further details contact: Scientific Secretariat Administrative Secretariat before and after the Conference H. P. Walliser, Ph. D. c/o Novartis Pharma AG K CH-4002 Basel Tel. (+41) Fax (+41) [email protected] Administrative Secretariat c/o AKM Congress Service P.O. Box CH-4005 Basel Tel. (+41) Fax (+41) [email protected] Table 1. Characteristics of HiPrep 26/10 Desalting. Matrix Sephadex G-25 Fine cross-linked dextran Particle mean diameter 90 µm Exclusion limit for proteins/peptides (M r ) Bed volume 53 ml Column dimensions (i.d.xh) 2.6 x 10 cm Recommended sample volume <15 ml Maximum flow rate 40 ml/min (450 cm/h) Recommended flow rate* 9-31 ml/min ( cm/h) Maximum pressure drop, Dp 1.5 bar (22 psi, 0.15 MPa) Chemical stability all common aqueous buffers ph stability 2-13 (long- and short-term) Storage 20% ethanol Avoid oxidizing agents *Water at room temperature Table 2. Run data for buffer exchange of 30 ml and 60 ml samples on HiPrep 26/10 Desalting columns connected in series. Columns Sample Sample preparation Sample loop Buffer 2 x HiPrep 26/10 Desalting in series (V T = 106 ml) for 30 ml sample or 4 x HiPrep 26/10 Desalting in series (V T = 212 ml) for 60 ml sample 30 ml or 60 ml Pichia pastoris culture supernatant containing a secreted recombinant protein filter through 0.45 µm filter Superloop TM 150 ml 0.1 M Tris TM, 0.15 M NaCl, 0.05% Tween TM 20, ph 7.6 Flow rates (30 ml) sample loading 12 ml/min elution 17 ml/min Flow rates (60 ml) sample loading 8 ml/min elution 11 ml/min Back pressure 4.8 bar (70 psi, 0.48 MPa) Fractions 5 ml Instrument ÄKTA TM explorer 100 Eluted sample 30 ml 35 ml (dilution factor, 1.2) 60 ml 70 ml (dilution factor, 1.2)

30 30 thirty Press release Expansion and growth in North America Official opening of the new facilities in Piscataway, NJ, USA. September, Amersham Pharmacia Biotech, one of the world's leading suppliers of enabling technology for medical research, is expanding its facility in Piscataway to become the corporate headquarters for the company's North American operations. Approximately 300 positions will be relocated to the Centennial Avenue site, for a total of 550 jobs at the facility, mostly in production, research and commercial operations. About $30 million in construction projects and site improvements are underway, including an engineering facility for BioProcess systems and columns. "We are pleased to make Piscataway the centre of excellence for our North American operations," said Philip G. Douglas, Ph.D., President, North America Holdings, Amersham Pharmacia Biotech. "We intend to be a good neighbour and a good corporate citizen. We want the community to benefit from our growth, and take pride in the work that we do here, which enables the discovery and development of life-saving medicines." The company's expansion in Piscataway, which involves relocating operations and people from Chicago, Cleveland and Milwaukee, is driven by its strategic location in the heart of the eastern seaboard's pharmaceutical industry. "We want to foster relationships and increase collaborations with our customers, the pharmaceutical and biotechnology companies and universities in the region," said Peter Coggins, Ph.D., President, Sales and Marketing, North America Region, Amersham Pharmacia Biotech. Useful reference works This thoroughly revised and updated edition of Protein Purification provides state-of-the-art coverage of chromatographic protein separation and characterization methods. Balancing theory, procedures and applications, it offers professionals and students in biochemistry, organic chemistry and analytical chemistry quick access to a wide range of important techniques. Protein Purification Edited by Jan Christer Janson and Lars Rydén. Published by Wiley-Liss, New York, 1998 ISBN: Amersham Pharmacia Biotech code no.:

31 thirty 31 Press release Q-One Biotech appoints Amersham Pharmacia Biotech KK as their exclusive distributor in Japan Q-One Biotech Ltd (Glasgow, Scotland, UK), a leading company worldwide in the field of contract biosafety testing, process validation and manufacture of gene therapy products, has formed a partnership with Amersham Pharmacia Biotech KK (Shinjuku, Tokyo) as of April 1st Although Q-One has been successfully building close contacts with the biopharmaceutical industry in Japan over many years, the appointment of Amersham Pharmacia Biotech KK as their exclusive distributor will provide those companies developing biotechnology products with a very high level of technical service and support. Many of Amersham Pharmacia Biotech's products and services are complimentary to those of Q-One's, offering the customer an integrated service. In the area of viral or TSE clearance studies of purification processes, Q-One has an international reputation, however Amersham Pharmacia Biotech is a world leader in the supply of purification systems and media and the validation of large and down-scaled processes. The design of efficient and cost-effective protein purification systems will be enhanced by this partnership, as biopharmaceutical companies will benefit from the combined expertise of the protein chemists and the virologists designing processes to satisfy the latest stringent regulatory requirements. This book offers a practical approach to developing an optimal chromatographic process, scaling it up, and adapting it to comply with requirements set by world-wide regulatory agencies. Dr Henke draws on his long professional experience of working with Sephadex TM LH-20. He describes the practical requirements for optimal chromatographic performance and experimental reproducibility. Process Chromatography: Optimization, Scale up and Validation G. Sofer and L. Hagel. Published by Academic Press, London, 1997 Amersham Pharmacia Biotech code no.: Preparative Gel Chromatography on Sephadex LH-20 Hans Henke, published by Hüthig, Heidelburg, 1995 ISBN: Amersham Pharmacia Biotech code no.:

32