Bioseparation Engineering (eBook)

I. Endo, S. Katoh, T. Nagamune, T. Yonemoto (Herausgeber)

eBook Download: PDF

2000 | 1. Auflage
240 Seiten
Elsevier Science (Verlag)
978-0-08-052815-1 (ISBN)

Infos

The bioseparation engineering of today includes downstream process engineering such as waste water, material and gas treatment. Taking this tendency into account, bioseparation engineers gathered in Japan as a special research group under the main theme of Recovery and Recycle of Resources to Protect the Global Environment.

The scope of this book is based on the conference, and deals not only with recent advances in bioseparation engineering in a narrow sence, but also the environmental engineering which includes waste water treatment and bioremediation. The contributors of this book cover many disciplines such as chemical engineering, analytical chemistry, biochemistry, and microbiology.

Bioseparation Engineering will stimulate young engineers and scientists who will develop bioseparation engineering further in the 21st century, and contribute to a world-wide attention to the global environment

---

The bioseparation engineering of today includes downstream process engineering such as waste water, material and gas treatment. Taking this tendency into account, bioseparation engineers gathered in Japan as a special research group under the main theme of "e;Recovery and Recycle of Resources to Protect the Global Environment"e;.The scope of this book is based on the conference, and deals not only with recent advances in bioseparation engineering in a narrow sence, but also the environmental engineering which includes waste water treatment and bioremediation. The contributors of this book cover many disciplines such as chemical engineering, analytical chemistry, biochemistry, and microbiology.Bioseparation Engineering will stimulate young engineers and scientists who will develop bioseparation engineering further in the 21st century, and contribute to a world-wide attention to the global environment

Inhalt

Front Cover 1
Bioseparation Engineering 4
Copyright Page 5
Contents 10
Preface 6
Acknowledgments 8
Chapter 1. Adsorption, Chromatography and Membrane Separations 14
Recent Advances in Membrane Technology that Could Improve Resource Recovery and Recycle: Fluid Mechanics, Surface Science and Bioaffinity 16
Stabilization of Target Protein during Bioseparation 22
Bioseparation of Natural Products 28
On-line Recovery of Large Molecules from Mixture Solution Using Semi-continuous Size Exclusion Chromatography 34
Dye Adsorption by Activated Carbon in Centrifugal Field 38
Formation and Structural Change of Cake during Crossflow Microfiltration of Microbial Cell Suspension Containing Fine Particles 42
Continuous Separation of Ternary Mixture of Amino Acids Using Rotating Annular Chromatography with Partial Recycle of Effluent 48
Mass Transfer Characteristics of a Perfusion-type Gel Analyzed by Shallow Bed Method 54
Fouling of Cheese Whey during Reverse Osmosis and Precipitation of Calcium Phosphate 60
Separation of Dead Cells from Culture Broth by Using Dielectrophoresis 66
Microcalorimetric Studies of Interactions between Proteins and Hydrophobic Ligands in Hydrophobic Interaction Chromatography: Effects of Ligand Chain Length, Density, and the Amount of Bound Protein 72
Membrane Phase Separation of Aqueous/Alcohol Biphase Mixture and Its Application for Enzyme Bioreactor 76
Microfabricated Structures for Bioseparation 82
Production of a Human IgM-type Antibody and Preparation of Combinatorial Library by Recombinant Saccharomyces cerevisiae 88
Dynamic Binding Performance of Large Biomolecules such as y-globulin, Viruses and Virus-like Particles on Various Chromatographic Supports 94
Effects of Swelling Pressure of Resin and Complex Formation with a Counter-ion on the Apparent Distribution Coefficient of a Saccharide onto a Cation-exchange Resin 100
Separation Behavior of Proteins near the Isoelectric Points in Electrostatic Interaction (Ion Exchange) Chromatography 106
Chapter 2. Refolding Processes for Protein 112
Large-scale Refolding of Therapeutic Proteins 114
Novel Method for Continuous Refolding of Protein with High Efficiency 120
Novel Protein Refolding by Reversed Micelles 126
Development of Efficient Protein Refolding Systems Using Chaperonins 132
Monitoring Structural Changes of Proteins on Solid Phase Using Surface Plasmon Resonance Sensor 138
Chapter 3. Partitioning and Extraction 144
Recent Advances in Reversed Micellar Techniques for Bioseparation 146
A Novel Method of Determining the Aggregation Behavior of Microemulsion Droplets 150
Preparation of Temperature-sensitive Antibody Fragments 156
Stability Enhancement of a-amylase by Supercritical Carbon Dioxide Pretreatment 162
Behavior of Monodispersed Oil-in Water Microsphere Formation Using Microchannel Emulsification Technique 168
Chapter 4. Bioseparation Engineering for Global Environment 174
Domestic Wastewater Treatment Using a Submerget Membrane Bioreactor 176
Biosorption of Heavy Metal Ion with Penicillin Biomass 182
Removal of Cadmium Ion 188
The Effects of Additives on Hydrolysis of Cellulose with Water under Pressures 194
Removal of Volatile Organic Compounds from Waste Gas in Packed Column with Immobilized Activated Sludge Gel Beads 200
Chapter 5. Industrial Separation Processes and Validations 206
Validation of Bioprocess Chromatography : Principles and Practices 208
Column Qualification in Process Ion-exchange Chromatography 214
Characterization of Phage Encoded Lysis Proteins and Its Applications for Cell Disruption 220
Recovery of Poly-b-hydroxybutyrate from Recombinant Escherichia coli by a Combined Biologi-chemical Method 226
Cleaning Liquid Consumption and Recycle of Biopharmaceutical Plant 232
Index of authors 238

Textauszug

Stabilization of target protein during bioseparation

X.-L. Fenga; Y.-T. Jinb; Z.-G. Sua    a National Laboratory of Biochemical Engineering, Institute of Chemical Metallurgy, Chinese Academy of Science, Beijing 100080, The People's Republic of China
b Laboratory of Biochemical Engineering, Dalian University of Technology, Dalian 116012, The People's Republic of China

Denaturation of target protein by various separation and purification steps contributes significant part to the total product loss in bioseparation. This report classifies the denaturation into four types including thermal denaturation, shear denaturation, solution denaturation and adsorption denaturation. For stabilization of target protein, three strategies are proposed including careful selection of unit operation to avoid detrimental action, process optimization to reduce the number of steps and the total processing time, and utilization of protective reagents such as PEG during bioseparation. It is important to understand the structure and property of the product to design the best bioseparation route.

1 INTRODUCTION

Low recovery is a major problem in production of pharmaceutical proteins. The loss of target protein can be classified into two aspects. The first one is physical loss in the flow stream, such as the leakage through an ultrafiltration membrane during concentration operation, the carry-away during a washing step in chromatography after loading, or even the residual left in the dead volume of a process device and the pipelines. This part of loss should not contribute to more than 15%, and is often controllable by proper process design and operation. The second loss is the denaturation of the target protein by various separation or purification steps. This part is significant, much more than 15%, and is difficult to control.

Any separation step in a bioprocess relies on its physical, chemical or biological action to distinct one or a group of proteins from the other. The product, or the target protein, has a limited stability undergoing the treatment. Even there is no change in the molecular weight or in the one dimensional structure, a minor alteration of the molecular conformation would result in loss of its biological activity. While molecular biologists are trying to construct artificial proteins that are more stable and functional, biochemical engineers are working hard in designing optimal separation routes to maintain the three dimensional integrity of the products and to achieve the desired purification during bioseparation [1].

2 AVOIDANCE OF DETRIMENTAL ACTION

In order to decrease the denaturation loss, care has to be exercised in choosing suitable separation methods to avoid detrimental actions, such as increasing temperature, excessive stirring, marked changes in pH, adding organic solvents and exposure to ultraviolet light. Table 1 lists the frequently used unit operations, its separation principles and possible damage to proteins. In general, protein denaturation in bioseparation can be classified into four categories, i.e. thermal denaturation, shear denaturation, solution denaturation and adsorption denaturation. Other denaturations such as those induced by high pressure and ultraviolet light are not common, and will not be discussed here.

Table 1

Denaturation of proteins in separation and purification

Unit operation

Separation principles

Damage to proteins

Cell disruption

Liquid shear, impingement, pressure change, hydrolysis of cell membrane & wall

Thermal denaturation, shear denaturation, solution denaturation

Aqueous twophase extraction

Partition in different phases driven by thermodynamics

Solution denaturation, shear denaturation

Centrifugation

Density difference

Thermal denaturation

Membrane filtration

Size difference

Shear denaturation, adsorption denaturation

Chromatography

Surface interaction, size difference

Adsorption denaturation, solution denaturation

Freeze drying

Volatility difference

solution denaturation

Thermal denaturation is caused by temperature increase, resulting in disorder of the three dimensional structure by breakage of the forces stabilizing the spatial conformation, such as hydrogen bonds, electrostatic and hydrophobic interactions. In mechanical cell disintegration such as homogenization and bead milling, part of the mechanical energy transferred to heat energy, increasing the temperature of the homogenate. For example, one passage through a homogenizer at 600 bars can increase the homogenate temperature by 2-5 °C depending on cell concentration and viscosity of the homogenate. Cooling is necessary for multiple passage of homogenization.

Shear denaturation is associated with high liquid flow rates. The mechanism is still unclear. Many observations have proved that protein may lost its activity in a high liquid shear field. For shear sensitive proteins, cross-flow microfiltration and ultrafiltration may cause denaturation due to high shear used for minimization of concentration polarization. Pumping is a process associated with liquid shear. Peristaltic pumps are normally regarded as mild operators and preferred choice for less contamination. However, studies have demonstrated that peristaltic pumps could denature proteins by generation of protein aggregates. The solution of serum albumin, in which aggregates had been removed, when being pumped again with a peristaltic pump, produced aggregates again. The pumping period and concentration of the protein determine the magnitude of aggregate formation [2].

For solution denaturation, several mechanisms may be involved, including protease hydrolysis, chemical hydrolysis, interaction with salts, surfactants, organic solvents etc.[3]. In fact these actions in solution may be going on all the time during bioseparation with varied degrees for different proteins, even the solution is in cold storage. When a separation requires addition of certain substances to the protein solution and process it under certain condition, denaturation by the substances present in the solution may occur. For example, chemical disruption of the cells requires addition of organic solvents, surfactants or chaotropic agents such as guanidine hydrochloride. These reagents break down cell membranes to release the intracellular protein. However, the released product is also under the attack of the reagents. Aqueous two-phase extraction in general is good for maintaining the activity of the protein, but the high concentration of salts and type of salts may affect the protein activity in salt- polymer system. Solution denaturation depends on the concentration of the solutes that denature the product. In freeze drying, much of the protein activity may be lost during freezing stage because water forms ice and solute concentrations are increased.

Adsorption denaturation happens on solid surface. Non-specific adsorption of a protein to the surface of a separation medium or any contacting materials of the process contributes to the denaturation significantly. Specific adsorption is a basis of chromatographic separation. For purification of pharmaceutical proteins, chromatographic steps must be involved. However, most chromatographic media are not totally selective with uniform adsorption pattern. Protein denaturation may take place on the surface of chromatographic media. Furthermore, elution of the target protein from the column requires specific solutions, such as those with extreme pH, high salt concentration or detergents. Considerable denaturation may occur during elution, especially in the case of affinity chromatography where the protein binds the ligand tightly, and harsh elution condition must be employed.

The four types of denaturation may happen simultaneously and interact with each other. For example, increasing temperature could not only cause thermal denaturation but also promote solution denaturation. High liquid shear also increases the temperature of the solution.

3 PROCESS OPTIMIZATION

It is understandable that the less the processing time and steps, the less the protein denaturation could be. In fact the rate of protein denaturation varies with different steps of bioseparation. As a general rule, protein should be processed as fast as possible. Inactivation of certain enzymes was found to be an exponential function of time [4] as

active=Co×e‐t/k

  (1)

where Cactive is the remaining activity after time t, C0 is the original activity, and k is a coefficient related to the protein structure and environment. Therefore, reduction of processing time is an obvious strategy for increasing protein recovery. During the last few years, process integration and optimization have been paid much attention. The goal is to make the process simpler and faster. Existing processes may be the duplicates of the protocols from molecular biology laboratories where the recombinant proteins were developed. Much of the concern at that time was placed on cloning and expression. As long as the protein can be purified, recovery is not the top priority. Such bioseparation process may be tedious, time consuming and high cost. It is the task for biochemical engineers to develop optimized process. In fact biochemical...

Produkt-Details

• Erscheint lt. Verlag: 17.3.2000
• Sprache: englisch
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Naturwissenschaften ► Biologie ► Ökologie / Naturschutz
• Themenwelt: Naturwissenschaften ► Chemie ► Analytische Chemie
• Themenwelt: Naturwissenschaften ► Physik / Astronomie ► Angewandte Physik
• Themenwelt: Technik ► Umwelttechnik / Biotechnologie
• ISBN-10: 0-08-052815-5 / 0080528155
• ISBN-13: 978-0-08-052815-1 / 9780080528151