Production and Purification of Recombinant Proteins
The growing therapeutic use of proteins has created an increasing need for practical and economical processing techniques. As a result, biotechnological production methods have advanced significantly over the last decade. Also, single-use production techn
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Production and Purification of Recombinant Proteins Farida Kadir, Paul Ives, Alfred Luitjens, and Emile van Corven
INTRODUCTION The growing therapeutic use of proteins has created an increasing need for practical and economical processing techniques. As a result, biotechnological production methods have advanced significantly over the last decade. Also, single-use production technology which has the potential to mitigate many of the economic and quality issues arising from manufacturing these products has evolved rapidly (Hodge 2004). When producing proteins for therapeutic use, a number of issues must be considered related to the manufacturing, purification, and characterization of the products. Biotechnological products for therapeutic use have to meet strict specifications especially when used via the parenteral route (Walter and Werner 1993). In this chapter several aspects of production (upstream processing) and purification (downstream processing) will be dealt with briefly. For further details, the reader is referred to the literature mentioned.
UPSTREAM PROCESSING ■ Expression
Systems
General Considerations Expression systems for proteins of therapeutic interest include both pro- and eukaryotic cells (bacteria, yeast, fungi, plants, insect cells, mammalian cells) and transF. Kadir, Ph.D. Postacamedic Education Pharmacists (POA), Bunnik, The Netherlands P. Ives, Ph.D. Manufacturing Department, SynCo Bio Partners BV, Amsterdam, The Netherlands Department of Process Development, Crucell, Leiden, The Netherlands A. Luitjens • E. van Corven, Ph.D. (*) Department of Process Development, Crucell, Leiden, The Netherlands e-mail: [email protected]
genic animals. The choice of a particular system will be determined to a large extent by the nature and origin of the desired protein, the intended use of the product, the amount needed, and the cost. In principle, any protein can be produced using genetically engineered organisms, but not every type of protein can be produced by every type of cell. In the majority of cases, the protein is foreign to the host cells that have to produce it, and although the translation of the genetic code can be performed by the cells, the posttranslation modifications of the protein might be different as compared to the original product. About 5 % of the proteome are thought to comprise enzymes performing over 200 types of posttranslation modifications of proteins (Walsh 2006). These modifications are species and/or cell-type specific. The metabolic pathways that lead to these modifications are genetically determined by the host cell. Thus, even if the cells are capable of producing the desired posttranslation modification, like glycosylation, still the resulting glycosylation pattern might be different from that of the native protein. Correct N-linked glycosylation of therapeutically relevant proteins is important for full biological activity, immunogenicity, stability, targeting, and pharmacokinetics. Prokaryotic cells, like bacteria, are sometimes capable of producing N-l
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