Bioseparations of Proteins

Unfolding/Folding and Validations

Ajit Sadana, Satinder Ahuja

• Publisher: Academic Press
• Year: 1997
• Print ISBN: 9780126140408
• eBook ISBN: 9780080528144
• Topic: Biological Sciences
• Subtopic: Biochemistry
• Index: Biological Sciences

This book covers the fundamentals of protein inactivation during bioseparation and the effect on protein processing. Bioseparation of Proteins is unique because it provides a background of the bioseparation processes, and it is the first book available to emphasize the influence of the different bioseparation processes on protein inactivation.Bioseparation of Proteins covers the extent, mechanisms of, and control of protein inactivation during these processes along with the subsequent and essential validation of these processes. The book focuses on the avoidance of protein (biologicalproduct) inactivation at each step in a bioprocess. It compares protein inactivation exhibited during the different bioseparation processes by different workers and provides a valuable framework for workers in different areas interested in bioseparations.Topics include separation and detection methods; estimates of protein inactivation and an analysis of this problem for different separation processes; strategies for avoiding inactivation; the molecular basis of surface activity and protein adsorption, process monitoring, and product validation techniques; and the economics of various bioseparation processes and quality control procedures.Key Features* Protein inactivation and other aspects of biological stability are critical to an effective bioseparation process; This book is a detailed and critical review of the available literature in an area that is essential to the effectiveness, validation, and economics of bioseparation processes for drugs and other biological products; Conveniently assembled under one cover, the survey of the literature and resulting perspective will greatly assist engineers and chemists in designingand improving their own processes; Key features of the text include: * detailed data on biological stability under various bioseparation conditions* extensive case studies from the literature on separation processes, validation, and economics* simplified analysis of protein refolding and inactivation mechanisms* consideration of adsorption theories and the effect of heterogeneity* coverage of both classical and novel bioseparation techniques, including chromatographic procedures

1

Introduction

Ajit Sadana

I INTRODUCTION

Advances in genetic engineering, rDNA technology, and cell fusion techniques have made it possible to produce proteins of interest; however, the technology has not kept pace with these advances in sufficient quantities. Baum (1987) emphasized the need to process biological products to a high degree of purity on a large scale. Diamond and Hsu (1989) emphasized that the separation procedures should be economical and biocompatible. The National Committee on Bioprocess Engineering (1993) identified the development of separation and purification strategies for biological products from dilute aqueous solutions as a critical need for obtaining specialty bioproducts and industrial chemicals. These dilute aqueous solutions are often obtained in processing biological materials from fermentation, plant cell culture, or whole plant material.

II THE NEED FOR BIOSEPARATION

Furthermore, with the ever-increasing emphasis on safety with regard to regulatory agency requirements and public awareness, Lilly (1992) correctly emphasized the increasing importance of product quality, and not just the amount of product produced during a process. Lilly (1992) emphasized that to maintain product quality undesirable posttranslational changes must be either minimized or prevented. These changes may occur during both upstream and downstream processing. Also, most proteins must be folded into a specific three-dimensional conformation to express their biological activities and specificity, which complicates the process of separating and purifying them. The high cost of separation and purification coupled with the difficulty of getting highly purified products prevents some biotechnological processes with applications in medicine, agriculture, and industry from becoming viable, cost effective, and successful.

People working in the industry realized this, and subsequently many of them got involved in protein separation and purification. As a result of their research, novel and imaginative techniques sprang up. Some researchers modified existing procedures such as chromatography, electrophoresis, and precipitation. As expected, not all the techniques developed have the potential to be applied extensively. Thus, new and novel bioseparation techniques are gradually being developed and analyzed for their effectiveness. Also, Wheelwright (1989) emphasizes that even though quite a few downstream processes are in operation, there is no definite and predictable method or algorithm that one may follow to design a bioseparation protocol for a specific protein or biological product. This author emphasizes that the number of processes available and the subtle differences that exist between the different proteins make the development of a generalized algorithm for the step-by-step design of a bioseparation protocol more difficult.

Even though the generalized development of a bioseparation protocol is seemingly difficult, simplistic guidelines coupled with invaluable hands-on experience should provide the next best approach. Hopefully, the availability of more information in this area with respect to all the aspects of the bioseparation protocol should move bioseparation from an art to a science. The chapters that follow are an attempt in this direction. Also, in general, protein purification techniques should be simple, easily scalable, continuous, low cost; and, of course, should not inactivate the protein. Also, continuous processes are not always desirable. For example, high-value therapeutic proteins are produced in a batch mode for different reasons, including cost and risk factors.

III CLASSIFICATION OF BIOSEPARATION STEPS

Cussler (1987) indicates that although a variety of bioseparation procedures exist, they can be classified into four distinct steps that include removal of insolubles, isolation of product, purification, and polishing. As is to be expected, a wide variety of bioseparation procedures are available. Because these processes contribute significantly to the cost of the product, Van Brunt (1985) emphasizes that the economic consequences of these processes must be carefully considered. Van Brunt (1985) indicates that bioseparation processes include, but are not limited to, cell disruption, centrifugation, chromatography, drying, evaporation, extraction, filtration, membrane separation, and precipitation. This author emphasizes that some of these processes are classical and their mechanisms of action are well documented in the literature. Some of the preceding processes still have to be proved, especially on the large-scale level.

The end product of interest to be obtained from these processes must meet varying, rather strict demands before it can be placed on the marketplace. For example, the product must be sterile; attain stringent quality requirements; and be free from detergents, endotoxins, proteases, etc. Curling (1985) indicates that a pure product should satisfy the demands of no immunogenic substances present, no unwanted biological activity present, no microbiological contamination, and no enzymatic activity present that is harmful to the product. For example, other proteins, modified proteins, nucleic acids, oligonucleotides, or nucleotides contribute to an immunogenic response. Enterotoxins and nonspecific activity (such as complement activation) contribute to unwanted biological activity.

In general, the end product quality requirements are largely dependent on the end use of the product. For therapeutic usage some of the requirements that are to be met include potency, identity, abnormal toxicity, nucleic acids, homogeneity, etc. (Desai, 1990). The bioseparation process or protocol that is utilized to separate the product must satisfy these requirements at the end. Huddleston et al. (1991) indicate that bioseparation processes are defined by the nature of the product and its application. For some cases a high degree of purity is required, whereas in others simply the absence of conflicting activity is sufficient. Huddleston et al. (1991) emphasize that during the initial bioseparation steps one attempts to maximize product yield even at the expense of retaining contaminants. These contaminants may be removed later using high-resolution fractionation processes. Furthermore, Huddleston et al. (1991) emphasize the compromise that is required in the bioseparation protocol during the harvesting, product release, clarification, enrichment, and fractionation stages. Besides, one has to be careful in the bioseparation protocol to maintain an adequate containment of any potentially hazardous by-products.

One will require a wide variety of steps in the bioseparation protocol to meet different demands on the quality of the end product. Harakas (1989), however, emphasized that one has to limit the number of steps; and one should get the most out of each step. Ideally, one should, if it is at all possible, try to restrict the bioseparation protocol to just two or three steps. Also, Harakas (1989) emphasized that one should attempt to obtain at least 90% of the product from each step. Thus, if we have two steps then the overall efficiency is 81%. If three steps are utilized, then the efficiency drops to about 73%. Note that three steps of efficiency of 80% each will eventually yield an overall efficiency of 51.2%. Thus, the need is to use as few steps as possible, and also to get as much as you can from each step.

This rapid decrease in overall efficiency has led different workers to integrate or to combine the different steps in the bioseparation protocol. This is also known as process intensification (Third International Conference on Separations for Biotechnology, 1994). Lyddiatt (1994) analyzed the use of fluidized diethylaminoethyl(DEAE)-Spherodex to combine the recovery of acidic protease with the fermentation of Yarrowia lipolytica cells. Also, Chang (1994) used expanded-bed adsorption for the direct extraction of glucose-6-phosphate dehydrogenase from modified yeast homogenate. This integration of steps may be either in the upstream process or in the downstream process.

Datar et al. (1993) have also recommended integration of unit operations. Hanson and Rouan (1994), too, have utilized the expanded-bed adsorption technique to directly recover secreted recombinant fusion protein from a crude fermenter broth. This was done without prior cell removal. The fusion protein was designed to exhibit a relatively low pI. This permitted the anionic exchange adsorption at pH 5.5. At this pH the other host proteins are not adsorbed. These authors obtained a 90% overall recovery using this procedure. Figure 1.1 shows the integration of the bioseparation steps using genetic design of this product. Nygren et al. (1995), too, emphasized that integrated processes may be utilized to yield biological products w...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright page
• Preface
• List of Examples
• 1: Introduction
• 2: Steps in Bioseparation Processes
• 3: High-Resolution Fractionation Processes
• 4: Interfacial Protein Adsorption and Inactivation During Bioseparation
• 5: Protein Inactivations During Chromatographic Methods of Separation
• 6: Protein Inactivations During Novel Bioseparation Techniques
• 7: Adsorption Influence on Bioseparation and Inactivation
• 8: Applications and Economics of Bioseparation
• 9: Protein Refolding and Inactivation During Bioseparation
• 10: Validation of the Production of Biological Products
• Index