Process Scale Liquid Chromatography

Edited by G. Subramanian

*j Weinheim - New York VCH W Base1 Cambridge - Tokyo

This Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left Blank

Process Scale Liquid Chromatography

Edited by G. Subramanian

0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim (Federal Republic of Germany), 1995

Distribution:

VCH, P. 0. Box 101161, D-69451 Weinheim (Federal Republic of Germany)

Switzerland: VCH, P. 0. Box. CH-4020 Basel (Switzerland)

United Kingdom and Ireland: VCH, 8 Wellington Court, Cambridge CBl 1HZ (United Kingdom)

USA and Canada: VCH, 220 East 23rd Street, New York, NY 100104606 (USA)

Japan: VCH, Eikow Building. 10-9 Hongo 1-chome, Bunkyo-ku. Tokyo 113 (Japan) 1

ISBN 3-527-28672-1

Process Scale Liquid Chromatography

Edited by G. Subramanian

*j Weinheim - New York VCH W Base1 Cambridge - Tokyo

Ganapathy Subramanian 60 B Jubilee Road Littlebourne Canterbury Kent CT 3 1TP. UK

This book was carefully produced. Nevertheless, authors. editor and publisher do not warrant the in- formation contained therein to be free of errors. Readers are advised to keep in mind that statcmcnts. data, illustrations, procedural details or other items may inadvertently be inaccurate.

Published jointly by VCH Verlagsgcsellschaft. Weinheim (Federal Republic of Germany) VCH Publishers. New York, NY (USA)

Editorial Director: Dr. Don Emerson, Dr. Steffen Pauly Production Manager: Claudia Gross1

Library of Congress Card No. applied for

British Library Cataloguing-in-Publication Data: A catalogue record for this book is available from the British Library

Die Dcutsche Bibliothek - CIP-Einheitsaufnahme Process scale liquid chromatography / ed. by G. Subramanian. - Weinheim ; New York ; BaSel ; Cambridge ;Tokyo : VCH, 1995

NE: Subramanian, Ganapathy [Hrsg.] ISBN 3-527-28672-1

0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim (Federal Republic of Germany). 1995

Printed on acid-free and low-chlorine paper

All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form -by photoprinting, microfilm, or any other means - nor transmitted or trans- lated into a machine language without written permission from the publishers. Registered names, trade- marks, etc. used in this book, even when not specifically marked as such. are not to be considered unpro- tected by law. Composition: K+V Fotosatz GmbH, D-64743 Beerfelden Printing: strauss offsetdruck GmbH, D-69509 Morlenbach Bookbinding: Wilhelm Osswald + Co., D-67433 Neustadt Printed in the Federal Republic of Germany

Preface

Preparative and process-scale liquid chromatography have gained considerable im- portance over the past two decades, not only as a research and development tool, but as a viable alternative to more traditional purification techniques in the production environment.

In recent years, there have been several advances in the development of matrix, designs, and systems, and in the understanding of theory, which have enabled liquid chromatography to be applied successfully in large-scale separations of biological molecules, thus making process-scale liquid chromatography a subject in its own right, but with its own problems as well.

Large-scale chromatography using different matrices for selective process separa- tions is carried out routinely in many areas of the bioprocessing industry. However, relatively little data is available in the scientific literature, for two main reasons. First- ly, many commercial processes involve proprietary technology, which precludes any opportunity for publication, and secondly, the cost of carrying out large-scale separations of a non-proprietary feedstock solely for academic purposes is often too high.

This book aims to provide a theoretical basis for the understanding and practical application of liquid chromatography in large-scale separations.

I am indebted to the contributors, who have shared their practical knowledge and experience. Each chapter represents an overview of its chosen topic. Chapter 1 de- scribes chromatography systems, designs and control systems for process-scale separations. The current state of theory in large-scale separation by liquid chroma- tography, for various applications, is discussed in Chapter 2, and alternative modes of operation of chromatographic columns in the process situation are presented in Chapter 3. The application of size-exclusion chromatography in process-scale purification of proteins is discussed in Chapter 4. Chapters 5 and 6 give an account of the application of polymeric media in process-scale separations and ion-exchange liquid chromatography in biochemical separations, respectively. Instrument design for industrial supercritical-fluid chromatography and its application in industrial separation, and the scaling up of supercritical-fluid chromatography to large-scale applications are described in Chapters 7 and 8. Affinity chromatography and its ap- plication in large-scale separations is reviewed in Chapter 9.

a

It is my hope that this book will bring the accumulated knowledge of process separations to scientists in industry, and that it will stimulate further progress in the field of process-scale liquid chromatography.

I wish to express my sincere thanks to Dr. Don Emerson and all his colleagues for their invaluable help.

Canterbury, Kent October 1994

G. Subramanian

Contents

1

1.1 1.2 1.2.1 1.2.2 1.2.3 1.2.4 1.2.5 1.2.6 1.2.7 1.2.8 1.3 1.4 1.4.1 1.4.2 1.4.3 1.4.4 1.4.5 1.4.6 1.4.7 1.5 1.5.1 1.5.2 1.5.3 1 S.4 1 S.5 1.5.6 1.6 1.6.1 1.6.2 1.6.3 1.7 1.8

Chromatography Systems - Design and Control

Fred Mann

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3 Material Compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electrical Standards

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Reliability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Serviceability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 System Design . . . . . . . . . . . . . . . . . . . . . 9 Component Selection 1 1 Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 Pumps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Valves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Pipework . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Temperature . . 20 UV/Visible Ads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Refractive Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 pH/Conductivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dedicated Controller . . . . . General Purpose Controller . . . . . . . . . . . . . . . . . . 24 Computer-Based Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

. .

VIII Contents

2 The Practical Application of Theory in Preparative Liquid Chromatography

Geoffrey B . Cox

2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Why Theory? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 How much Theory? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Single Solutes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1 Mass Overload . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1.1 A Simple Model: Single Component which Follows a Langmuir

Isotherm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1.2 Computer Simulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2 Volume Overload . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Multiple Solutes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.1 Computer Simulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.2 The Effects of Column Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Optimisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.1 Production Rate Optimisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.2 Cost Optimisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.2.1 Laboratory Scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.2.2 Production Scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.3 Practical Optimisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Appendix 1 . Calculation of Column Saturation Capacity . . . . . . . . . . . . . . . . Appendix 2 . Mathematical Models for Preparative Chromatography . . . . . .

Mass-Balance Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Craig Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3 Alternative Modes of Operation of Chromatography Columns in the Process Situation

Derek A . Hill

3.1 3.2 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 3.3

3.4

Process Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Elution Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Displacement Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Frontal Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Operating Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Miscellaneous Operating Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Alternative Chromatographic Modes and Techniques . . . . . . . . . . . . .

The Use of Alternative Modes and Techniques in Process Situations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

33 33 34 34 35

38 41 42 44 46 50 54 56 58 58 59 61 63

64 65 65 67

71 73 73 74 75 76 77

78 80

Con tents IX .

4 Process Scale Size Exclusion Chromatography

Jan-Christer Janson

4.1 4.2 4.3 4.3.1 4.3.2 4.3.3 4.3.4 4.4 4.5 4.6 4.6.1 4.6.2 4.6.3 4.7 4.8

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Separation Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Column Packing Materials for Process Scale SEC . . . . . . . . . . . . . . . 85 Dextran Gels and Polyacrylamide Gels . . . . . . . . . . . . . . . . . . . . . . . . . 85 Agarose Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Composite Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 The Choice of Separation Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Adsorption Effects of SEC Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 The Eluent in SEC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Practices of Process Scale SEC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 Column Dimension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 Gel Preparation and Column Packing . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Feed Stock Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatographic Productivity in SEC . . . . . . . . . . . . . . . . . . . . . . . . . . Strategy for Scaling-up of SEC

93 96 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5 Polymers and their Application in Liquid Chromatography

Linda L . Lloyd and John E Kennedy

5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 5.2 The Polymer Network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 5.3 Manufacturing Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 5.4 Types of Polymeric Matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 5.4.1 Synthetic Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 5.4.1.1 Polystyrene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 5.4.1.2 Polyacrylamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 5.4.1.3 Polymethacrylate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 5.4.1.4 Miscellaneous Synthetic Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 5.4.2 Natural Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 5.4.2.1 Dextran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 5.4.2.2 Agarose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 5.4.2.3 Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 5.4.3 Composite Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.4.3.2 Pellicular Supports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 5.4.3.3 Core Shell Grafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 5.4.3.4 Pore Matrix Composites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 5.4.3.5 Interpenetrating Networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 5.5 Polymer Physico-chemico Characteristics . . . . . . . . . . . . . . . . . . . . . . . 109 5.5.1 Particle Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 5.5.2 Pore Size and Pore Size Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

5.4.3.1 Surface Coatings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

X Con tents

5.5.3 Surface Area . . . . . . . . . . . . . . . . . . . . . . . . . 111 5.5.4 Mechanical Rigidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 5.5.5 Column Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 5.5.6 Eluent Compatibility and Solvent Strength . . . . . . . . . . . . . . . . . . . . . . 116 5.5.7 Activation and Functionalisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 5.5.7.1 Polystyrene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 5.5.7.2 Polyacrylamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 5.5.7.3 Polymethacrylate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 5.5.7.4 Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 5.6 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 5.6.1 Size Exclusion Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 5.6.2 Reversed Phase Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 5.6.3 Hydrophobic Interaction Chromatography ...................... 121 5.6.4 Ion Exchange Fractionations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 5.6.5 Affinity Supports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 5.6.6 Chiral Separations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 5.6.7 Hydrophilic Interaction Chromatography . . . . . . . . . . . . . . . . . . . . . . . 124 5.6.8 High Speed Separations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 5.7 Practical Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 5.7.1 Choice of Adsorbent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 5.7.2 Chemical Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 5.7.3 Fouling and Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 5.7.4 Recovery of Mass and Biological Activity ....................... 127 5.8 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

. . . . . . . . . . . . . . . . . . . .

6 Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography

Peter R . Levison

6.1 6.2 6.3 6.4

6.4.1 6.4.2 6.4.3 6.4.4 6.5

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Principles of Ion-Exchange Chromatography .................... 132 Throughput . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography ............................................. 236

142 146 247

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

Chromatography of Hen Egg-White Proteins .................... 137 Chromatography of Goat Serum Proteins . . . . . . . . . . . . . . . . . . . . . . . Chromatography of a Monoclonal Antibody Chromatography of DNA-Modifying Enzymes . . . . . . . . . . . . . . . . . . .

....................

Contents XI .

7

7.1 7.2 7.3 7.3.1 7.3.2 7.3.3 7.3.4 7.4 7.4.1 7.4.2 7.4.3 7.5 7.6

8

8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9

8.10

8.11

8.12 8.13 8.14

Instrumental Design and Separation in Large Scale Industrial Supercritical Fluid Chromatography

Pascal Jusforgues

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 Principle. Advantages and Drawbacks . . . . . . . . . . . . . . . . . . . . . . . . . . 153 Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 Pumping System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 Chromatographic Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 Fraction Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 Eluent Recycling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 Separation Costs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 Why PS-SFC is Expensive . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 Why PS-SFC is Cheap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 Purification Costs Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 Applications: SFC vs HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

Scaling-up of Supercritical Fluid Chromatography to Large-Scale Applications

Christopher D . Bevan and Christopher J: Mellish

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 Supercritical Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 Choice of Supercritical Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 The Scaling-up Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 The History of Preparative SFC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Safety Considerations . The Column Shield Jacket . . . . . . . . . . . . . 171 The Basic Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 Loading and Injection of Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 Design and Construction of the Sample Introduction Pressure Vessel (SIPV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 Collection of Fractions from the Preparative Supercritical Fluid Chromatograph . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 High Pressure Trapping with Subsequent Recovery by Solidification of the Carbon Dioxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 Development of Large Scale Commercial Systems . . . . . . . . . . . . . . . . 186 Detection of Solutes in Preparative SFC ........................ 188 Recent Developments in SFC and SFE ......................... 189

XI1 Contents

9 Affinity Chromatography and its Applications in Large-Scale Separations

Christopher R . Goward

9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193 9.2 Support Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194 9.3 Important Features of a Ligand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 9.3.1 Coupling of a Ligand to the Support Matrix . . . . . . . . . . . . . . . . . . . . 197 9.3.2 Activation of the Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198 9.3.3 Capacity of the Adsorbent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198 9.3.4 Ligand Leakage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 9.3.5 Triazine Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 9.4 Process Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

9.5 chromatography Column and Other Equipment . . . . . . . . . . . . . . . . . 202 9.6 Process Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 9.7 Chromatography Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 9.7.1 Adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 9.7.2 Washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204 9.7.3 Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 9.7.4 Selective Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 9.7.5 Non-selective Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 9.7.6 Flow Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 9.8 Cleaning and Storage of Adsorbents . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 9.9 Protein Engineering Applied to Protein Purification . . . . . . . . . . . . . 208 9.9.1 Release of the Affinity Tail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208 9.9.2 Examples of the Use of Affinity Tails .......................... 209 9.10 Examples of Some Large-Scale Affinity Methods . . . . . . . . . . . . . . . . 210

9.10.2 Streptavidin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 9.10.3 Glucokinase and Glycerokinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 9.10.4 Human Serum Albumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 9.10.5 Immunoaffinity Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212

9.4.1 Scale Up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

9.10.1 Protein G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

Contributors

Christopher D. Bevan Glaxo Group Research Ltd Structural Chemistry Department Greenford Road Greenford Middlesex UB6 OHE United Kingdom (Chapter 8)

Geoffrey B. Cox Prochrom Chemin des Blanches-Terres BP 9 F-54250 Champigneulles France (Chapter 2)

Christopher R. Goward Camar Portondown Salisbury SP4 OJG United Kingdom (Chapter 9)

Derek A. Hill The Wellcome Foundation Ltd Temple Hill Dartford DAI 5AH Kent United Kingdom (Chapter 3 )

Jan-Christer Janson Pharmacia Bioprocess Technology AB S-75 182 Uppsala Sweden (Chapter 4 )

Pascal Jusforgues Prochrom Chemin des BlanchesTerres BP 9 F-54250 Champigneulles France (Chapter 7)

John F. Kennedy Birmingham Carbohydrate and Protein Technology Group Research Laboratory for the Chemistry of Bioactive Proteins and Carbohydrates School of Chemistry University of Birmingham Edgebaston Birmingham, B15 2TT United Kingdom (Chapter 5 )

XIV Contributors

Peter R. Levison Whatman International Ltd Springfield Mill Maidstone ME14 2LE Kent United Kingdom (Chapter 6 )

Linda L. Lloyd Chembiotech Ltd Institute of Research and Development University of Birmingham Research Park Vincent Drive Birmingham B15 2SQ United Kingdom (Chapter 5 )

Fred A. Mann Amicon Ltd Upper Mill Stonehouse Gloucestershire GLlO 2BJ United Kingdom (Chapter I )

Christopher J. Mellish Glaxo Group Research Ltd Bioengineering Unit Greenford Road Green ford Middlesex UB6 OHE United Kingdom (Chapter 8)

List of Symbols and Abbreviations

A B C ci(k, I ) c m

c m , i

C S

cs, i

D m

dP

Ki

H K

k' kb L Lf N

Parameter in Knox Eqn (11); relates to packed bed Parameter in Knox Eqn; relates to diffusion in mobile phase Parameter in Knox Eqn; relates to mass transfer between phases Mobile phase concentration at the kth time step and Ith distance step Mobile phase concentration of solute Mobile phase concentration of solute i Stationary phase concentration of solute Stationary phase concentration of solute i Diffusion coefficient of solute in the mobile phase Particle diameter of packing material Height equivalent to a theoretical plate (L/N) Distribution coefficient between the mobile and stationary phases Distribution coefficient of solute i between the mobile and stationary phases Capacity factor in non-linear range of the isotherm Capacity factor in linear range of the isotherm Column length Loading factor (w,/ws) Column efficiency (no. of plates) for peak in non-linear range of the isotherm Column efficiency for peak in linear range of the isotherm Number of Craig stages Operating pressure Stationary phase concentration at the kth time step and Ith distance step Time Elapsed time per transfer in Craig simulation Elution time of non-retained peak Linear flow velocity Volume of mobile phase in the column (interstitial+ pore volume) Volume of stationary phase in column Mass of sample injected Column saturation capacity Longitudinal distance in column

XVI List of Symbols and Abbreviations

At Az @ Phase ratio VJV, @ Column resistance parameter rl Solvent viscosity V Reduced flow velocity (ud,/D,)

Time step in finite difference equation (A2.3); [ = 2H(1 +k’) /u] Distance step in finite difference equation; [ = HI

1 Chromatography Systems - Design and Control

Fred Mann

1.1 Introduction

‘Anything formed of parts placed together’, ‘a set of things considered as a con- nected whole’ is the dictionary definition of a system. A liquid chromatography system is thus defined as parts or components that are connected together to allow the process of liquid chromatography to take place.

All chromatography systems are basically the same (Fig. 1-1) whether they are used for analytical, small scale preparative or process use. The main components are com- mon and consist of:

- the stationary phase or matrix - the column to contain the matrix - a pump to push mobile phase through the column - means for selecting or mixing different solvents to produce gradients,

either step or linear - sample injection - detection on the column outlet - fraction collection - control and/or data collection

The particular use, however, for which a chromatography system is required, will in- fluence the relative importance and requirements of the individual components.

In analytical systems for instance, the objective is to identify components present in a small sample volume; consequently these systems must be able to accommodate highly efficient columns containing very small particle diameter packings producing high column back pressures. Minimal volume in pipework, valves, and detector flow cells are required and a large emphasis is placed on data handling with automatic calculation of peak areas for concentration determination. There is no requirement for a fraction collector.

In preparative systems, on the other hand, where the objective is to obtain purified components of the sample, fraction collection is a necessary requirement. However, preparative systems also differ depending on their use. In process development where the system may be used in the investigation and development of many different purification problems, flexibility is paramount, with the ability to operate with dif-

2 I Chromatography Systems - Design and Control

Soivent reservoirs

D

C

B

A

Column

Controller Recorder

I I I I

De tec to r

Fraction vessels

Fig. 1-1. Basic chromatograph components.

ferent media, solvents, columns, and detection requirements. In production where a system is dedicated to a single use, flexibility is no longer required and reliability will be the most important criteria.

In terms of control even the simplest flash chromatography separation is usually in reality, under very sophisticated control; that is the direct manual control of the operator who performs and coordinates all the functions of: solvent selection, mix- ing, solvent and sample addition to the column, visual monitoring of the flow or movement of coloured bands and collection of the different separated components in suitable receptacles.

However, by utilizing instrumentation to monitor the state of the system together with automation of valve switching and pump control, the direct operator involve- ment can be reduced, thus lowering labour cost, and increasing reproducibility and reliability.

1.2 System Requirements

Before entering into the detailed design of systems it is first beneficial to consider the overall requirements the system must satisfy, namely:

1.2 System Requirements 3

- functionality - material compatibility - pressure requirements - electrical standards - hygiene - control/automation - reliability - serviceability

1.2.1 Functionality

Chromatography separations are based not on the system but on the sample interac- tion with the stationary and mobile phases. If, however, the potential of this interac- tion is to be realized the system must not adversely affect the process.

In reality the effect of the system must inevitably be to reduce the efficiency of the separation, for instance by sample dilution in the pipework. The challenge in designing a system is to reduce this negative impact to a minimum.

In considering the functionality, therefore, it is necessary to consider not only the number of required solvent inlets, fraction outlets, pump and valve types, sensors, detectors, etc., but also the pipework size and configuration to ensure that they are optimal for the required flow and that dead legs and dilution zones are kept to a min- imum.

1.2.2 Material Compatibility

Selection of materials should be such that no problems can arise from adsorption to, or leaching from, components within the system. Materials must be compatible with all solutions used in the process, including those used for regeneration, clean- ing, and storage [I]. Particular attention must be paid to the use of plastics or elastomeric seals with organic solvents. The effect of solvents is dependent on con- centration, temperature and contact time. Attack may result in softening and dissolv- ing of the polymer and/or leaching of plasticizers or other components. For these reasons HPLC systems invariably use only stainless steel and PTFE as construction materials.

It is particularly important to ensure that plastic or elastomeric materials selected for incorporation into systems destined for use in pharmaceutical production pro- cesses are acceptable and will satisfy any regulatory requirements (eg, US Food and Drug Administration (FDA) [2], US Pharmacopeia [3]).

In the case of aqueous systems where halide ions are present, stainless steel of at least 3 16L grade is required, and in the case of high concentrations of chloride even more resistant grades may be specified. Alternatively, plastic may be used in place of steel.

4 I Chromatography Systems - Design and Control

Attention should also be given not only to materials in direct contact with the pro- cess stream but also to external materials used for manufacture of frames and cabinets. Many chromatographs used for pilot or production purposes are located in environments promoting corrosion (eg, wet areas) and appropriate specification of these external materials is just as important as that applied to process pipework.

1.2.3 Pressure

It is necessary to ensure that not only are all components within the system ap- propriately rated for the maximum operating pressure of the system, but that the design of the system adequately covers the pressure drop in the column and the asso- ciated pipework and valving at the maximum flowrate required.

The major contribution to pressure drop in the system is invariably the packed col- umn bed. Information on this can be derived from the early process development trials and from the matrix manufacturer. Caution should be exercised to ensure that the data relates to performance of the matrix in the column diameter proposed. With rigid particle packings (eg, Silica) data from small-diameter columns (25 - 50 mm diameter) can readily be extrapolated to larger diameter columns.

In the case of soft or deformable matrices this may not be the case. With these matrices in small diameter columns, the bed is supported by the wall of the column, the effect of which is lost as the diameter of the column is increased (>200mm diameter). This leads to greater than expected pressure drops in larger columns. In some cases the matrix may be so deformable that required flow rates cannot be achieved in large-diameter columns due to the compressibility of the matrix.

Pressure drop is related to flowrate and this relationship is particularly important when designing a system with a wide flowrate range. The pressure/flow relationship of a packed bed is linear, whereas with pipework it is a square factor where a doubl- ing in flowrate will result in a four-fold increase in pressure drop. Consequently, although in order to minimize dilution within the system at low flowrate, it is desirable to use small bore tubing and valves, this may produce at high flowrates an undesirably high pressure drop requiring a compromise between minimum system volume and pressure drop.

Pressure drop after the column should be given special care as the column inlet pressure, to which the column must be designed and built, will be the sum of both the packed bed/column pressure drop and that of the post column pipework and valving. Whereas increasing the pressure rating of a small analytical column is rela- tively inexpensive, increasing the pressure ratings of large scale production columns can be considerable, due to the need for greater material thicknesses and differing designs to cope with, in particular, the higher end loadings on the larger columns as the pressure increases.

Implications of pressure vessel regulations also need to be considered, especially with respect to the column. Requirements differ between countries and is usually dependent on a combination of pressure and volume. For instance the A D Merkblatt regulations [4] in Germany stipulate more stringent requirements for design and

1.2 System Requirements 5

testing of a column if P (bar) x V (litres) is > 200. In the US many states classify larger chromatography columns as unfired pressure vessels, necessitating that they be designed, built and tested in accordance with the American Society of Mechanical Engineers (ASME) regulations [ 5 ] .

1.2.4 Electrical Standards

Electrical design and manufacture also needs to conform to appropriate standards. In the past each country had its own standards and regulations but there are now unified European standards under the auspices of CENELEC (European Committee For Electrotechnical Standardization) which covers the following countries: Austria, Belgium, Denmark, Finland, France, Germany, Greece, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the UK.

Each country still tends to maintain local standards classifications but these are directly equivalent to the European Norms. For instance an electrical design and manufacture applicable to process chromatographs is British Standard BS 2771 [6] (Electrical Equipment of Industrial Machines) which is equivalent to European Norm EN 6024.

In addition the nature of the environment in which chromatographs are installed, and their use with the potential for liquid leaks or wash down will necessitate that enclosures meet the appropriate standards for dust and water protection (Table 1-1). Most installations will utilize IP54 protection.

The use of organic solvents can mean that equipment needs to be designed and built to comply with standards for explosion proofing enabling it to operate in a hazardous area. Sometimes the chromatograph itself does not use flammable sol- vents but is installed in a hazardous area where flammable solvents are being used for other processes. In this case the chromatograph will still need to be built to com- ply with explosion proofing regulations. Individual country regulations have also

Table 1-1. Summary of IP protection numbers.

IP codes: ingress protection

First numeral Second numeral

0 No protection 1 Objects >50mm 2 Objects >12mm 3 Objects >2.5 mm 4 Objects >l.Omm 5 Dust protected 6 Dust tight

0 No protection I Vertically dripping water 2 75 to 90" angled dripping water 3 Splashed water 4 Sprayed water 5 Water jets 6 Heavy seas 7 Effects of immersion 8 Indefinite immersion

Protection against liquids Protection against solid bodies

6 1 Chromatography Systems - Design and Control

been standardized by CENELEC for European countries. In the US appropriate sec- tions of the NEC (National Electrical Code) apply.

1.2.5 Hygiene

The purification of sample feedstocks derived from micro-organisms or natural products, coupled with the use of mobile phases containing buffers and other com- ponents designed to maintain biological activity, can present ideal conditions for contamination of the system and proliferation by unwanted micro-organisms and the generation of pyrogens.

In these circumstances, effective cleaning, and if necessary sterilization is required if a product of the required purity is to be obtained. This problem is encountered more in low or medium pressure systems than in HPLC. In the latter, solvents, matrices, and sample feedstocks are less conducive to microbial growth.

In low and medium pressure chromatography, process hygiene is not only impor- tant in terms of preventing contamination of the product but also in prolonging the life of the stationary phase; the polymeric gel matrices commonly used being suscep- tible to bacterial degradation. In such cases the systems are cleaned in place (CIP) with strong alkaline or other appropriate cleaning or sanitizing solutions. For this to be effective the cleaning solution must be carried effectively throughout the entire system.

Sanitary design aims to meet the above requirements by ensuring that there are no unswept volumes within the flow path that would provide opportunities for micro- organisms to be harboured. No standards per se exist for sanitary designs of chromatographs but well established principles are utilized from other process ap- plications, for instance the dairy industry (ie, the American 3-A standards developed by the US Dairy Industry).

‘Tri-clamp’ style pipework fittings are preferred in place of threaded or ferrule- type connectors, as the face to face seal with flush fitting gasket does not provide crevices for microbial growth. In contrast, ferrule fittings invariably provide a ‘dead area’ between the tube and outer fitting in front of the ferrule. Similarly in the case of valves, diaphragm types are preferred as the design permits free flow across the whole internal surface, in contrast to ball valves where not only can there be a con- taminating ‘plug’ within the ball itself, but the area between the ball and packing seal may form a crevice for microbial growth.

In the design of sanitary systems attention has increasingly been focused, not only on the need for effectively flushed fittings and connections, but also on the surface of the pipework, valve or column itself, being non-conducive to bacterial or fungal attachment and growth. The smoother a surface the easier it is to clean and the less likely it is to harbour micro-organisms.

Stainless steel is traditionally polished mechanically using an abrasive polishing pad or mop with an abrasive paste. A coarse abrasive is used first to take out major imperfections with successively finer grades being used to obtain a smoother surface. The surface finish is often specified in terms of the final abrasive grade used, for in-

1.2 System Requirements 7

stance, a 180 grit finish. The higher the grit number the smoother the finish. Although grit number is very often used to describe the finish, variation may occur between different sources of the same grit number and also be dependent on how worn the abrasive is.

A more accurate comparison can be made by actual measurement of the surface texture of the steel. A stylus type instrument is used which is moved across the sur- face and produces a trace of the surface profile, together with a ‘Roughness Average’ (Ra) reading. Comparison of the Ra measurements still requires caution as correct setting of the measuring instrument (ie, cutoff) relative to the surface being measured is critical if representative results are to be achieved. Table 1-2 shows a comparison of typical Ra values for different grit finishes.

Table 1-2. Surface finish.

Grit finish Typical Ra“

Pm Micro inch

120 Grit 180 Grit 240 Grit 320 Grit

0.8-1.2 0.4-0.8 0.3 -0.4 0.15-0.3

31-47 15-31 11-15 6-11

a Ra is the roughness average. Ra is also known as the arithmetic average (AA) and centreline average (CLA). It is the arithmetic average of the absolute values of the measured profile height deviations taken within the sampling length and measured from the graphical centreline.

The surface of stainless steel, even if highly polished mechanically, is in fact not smooth but consists of a series of peaks and troughs. In fact the very act of polishing, being an abrasive process, actually increases the number of peaks and troughs and thus the actual surface area available for bacterial attachment.

In addition the polishing action tends to ‘bend over’ the tops of the peaks thus trapping polishing abrasive or other dirt within the troughs which can make subse- quent cleaning difficult. Increasingly electropolishing is being used as a final polishing step in addition to, or even as, a replacement for traditional mechanical polishing.

Electropolishing is in effect the reverse of electroplating. It is an electrolytic pro- cess with the item to be polished made anodic in a strongly acidic electrolyte and positioned adjacent to a formed cathode plate. A high anode surface current pro- gressively and preferentially dissolves the metal at the peaks of the surface. The peak to trough height is reduced and a smoother brighter surface with a lower total surface area results. The benefit of electropolishing in reducing the growth of bacteria on the steel surface has been demonstrated [7].

Stainless steel is utilized because of its high resistance to corrosion which is a result of the thin passive surface oxide film which will form naturally in the air. However, this passive film will only occur on clean surfaces. If areas of the surface are covered