Centrifugal Separations in Biotechnology

Wallace Woon-Fong Leung

• Publisher: Elsevier
• Year: 2020
• eBook ISBN: 9780081026359
• Edition: 2
• Topic: Technology & Engineering
• Subtopic: Chemical & Biochemical Engineering
• Index: Technology & Engineering

Centrifugal Separations in Biotechnology, Second Edition, is the only book on the market devoted to centrifugal separation in biotechnology. Key topics covered include a full introduction to centrifugation, sedimentation and separation; detailed coverage of centrifuge types, including batch and semi-batch centrifuges, disk-stack and tubular decanter centrifuges; methods for increasing solids concentration; laboratory and pilot testing of centrifuges; selection and sizing centrifuges; scale-up of equipment, performance prediction and analysis of test results using numerical simulation.Centrifugal Separations in Biotechnology, Second Edition, provides guidance on troubleshooting and optimizing centrifuges, and then goes on to explore the commercial applications of centrifuges in biotechnology. It gives detailed process information and data to assist in the development of particular processes from existing systems. It is of value to professionals in the chemical, bioprocess, and biotech sectors, and all those concerned with bioseparation, bioprocessing, unit-operations and process engineering.- Provides a comprehensive guide to centrifuges, their optimal development, and their operation in the biotechnology industry- Updated throughout based on developments in industrial applications and advances in our understanding of centrifugal separations in biotechnology- Discusses applications for the separation of proteins, DNA, mitochondria, ribosomes, lysosomes and other cellular elements- Includes new sections on use of optimal polymer dosage in waste treatment, new centrifuge designs for applications in algae processing, biopharma, and more

1

Introduction

Abstract

In this chapter, some important applications in biotechnology, such as manufacturing of drug substances purely from biologically derived products, have been presented. These processes are discussed generically so that they can be applicable for various situations. Also, it is important to understand that centrifugation should be partnered closely with other process equipment, both upstream and downstream, to make the entire process work using an integrated approach. Various separation metrics for centrifugation are discussed, including protein yield, centrate suspended solids, throughput, and cell viability. These subjects will be taken up in greater detail in the text. The organization layout of this book has been outlined for the need of the reader.

Keywords

Recombinant; protein; extracellular; intracellular; mammalian; yeast; bacteria; E. coli; separation; centrifugation; membrane; downstream processing; monoclonal antibody

1.1 Introduction

Biotechnology has revolutionized our life in the 20th century [1]. Its impact is only now being felt from engineering food [2–4], engineering and delivering drugs [5,6], to engineering consumable products. Without doubt it will continue to influence our daily lives for years to come. One of the many successful examples is that drugs, such as monoclonal antibodies (mAbs) and some basic drug substances (i.e., the building block for various drugs), can now be manufactured and formulated from bioreaction. One of the commonly used methods in biotechnology is the recombinant DNA technique [7–9]. A desired gene is isolated from one organism, and this is inserted into a small piece of carrier DNA called a vector. It is highly desirable that the recombined DNA (vector plus gene) can propagate in a similar or unrelated host/recipient cell.

The mammalian cell, such as the Chinese Hamster Ovary cell, is a popular host cell. Fig. 1.1 shows a schematic of an animal cell which is very similar to that of a mammalian cell. A characteristic size of the mammalian cell is about 10–20 µm. Unlike a plant cell, there is no cell wall for animal and mammalian cells, so they rely on a plasma membrane to keep the intracellular contents intact. High shear stress acting on the cell can rupture the fragile membrane releasing the intracellular material.

Figure 1.1 Animal cell schematic showing plasma membrane.

Yeast (see schematic in Fig. 1.2), in eukaryotic single-celled microorganisms classified as a member of fungus kingdom, has been commonly used as a host cell in the recombinant DNA process, the knowledge and experience of which we have gained from the brewery industry. Unlike a mammalian cell, the yeast cell has a strong cell wall. Yeast cells are smaller than mammalian cells and are typically between 7 and 10 µm. Some common yeast hosts include, Saccharomyces cerevisiae (referred commonly as baker yeast) and Pichia pastoris.

Figure 1.2 Yeast cell schematic showing both cell wall and membrane.

Bacteria, such as Escherichia coli (hereafter abbreviated as E. coli) and Bacillus subtilis, have been used as host cells for the recombinant DNA technique. A schematic of E. coli bacteria cell is shown in Fig. 1.3. Again, E. coli has a sturdy cell wall with both an outer membrane and an inner membrane. E. coli is typically elongated with a dimension of 3 µm long by 1 µm width. Therapeutic protein can be “expressed” by these host cells or organisms with the recombinant DNA. The protein of interest may remain in the cell (intracellular) or be secreted to the exterior of the cell (extracellular). The aforementioned biosynthesis provides more engineering flexibility, specificity, versatility, reliability, and cost-effectiveness.

Figure 1.3 Escherichia coli cell schematic.

Therapeutic proteins are quite diverse in the application treatments, such as human insulin for diabetes, erythropoietin for anemia and chronic renal failure, interferon-beta and gamma for cancer, DNase for pulmonary treatment, vaccines for hepatitis B, interleukin-2 for AIDS, prourokinase for heart attacks, and tissue plasminogen activator (enzyme) for strokes. Therapeutic proteins are present in many different kinds of mAbs.

mAbs are antibodies that are identical, because they are produced by one type of immune cells, and they are all copies or clones of a single parent cell. mAbs are first produced by Kohler and Milstein in 1975 [10], for which they were awarded the Nobel Prize in Physiology or Medicine in 1984. By virtue of the mAb being identical copies produced by one type of immune cells, they have a high specificity for their targets. mAb has been used in diagnosis. There are over 100 different diagnostic products available in the world that are mAb [11]. mAb is also used for therapeutics. For example, mAb has been a popular antibody made in the laboratory used for cancer treatment where the antibody is designed to attach as a label to their counterpart protein (antigen) on a specific cancer cell so that immune cells can spot and attack the cancer cells. As an example, the mAb known commercially as alemtuzumab drug (note all mAb drugs have the last three alphabets labeled as “mab,” which distinguishes them being mAb-based drugs) can target at the antigen CD52 found on the cancer cells that causes chronic lymphocytic leukemia [12]. As antigens are discovered to be linked to more specific cancers, more mAbs have also been developed for cancer treatment. Some mAbs work better on certain cancers than others. mAb can also attach to the antigen on breast cancer cells blocking the growth of breast cancer cells.

Cancer cells can “turn off the switch” of immune cells to avoid being attacked by the immune system in our bodies. Inhibitors, or commonly known as checkpoints, are mAb produced by the recombinant protein process, that inhibit the protein secreted from the cancer cells in “fooling” the immune cells, thereby allowing the immune cells to carry out their normal functions. As an example, PD-1 is a protein on the immune T-cells. The immune T-cells are normally in a switch-off condition because PD-1 has been attached by their counterpart PD-L1, another protein that both normal cells and cancer cells have. In other words, they have been switched off. Some cancer cells have an abundant PD-L1 that is used to attach to the PD-1 of the immune T-cells thereby evading being attacked. On the other hand, mAb can target at either PD-1 or PD-L1 and inhibit their binding, thereby allowing the immune cells to attack the cancer cells. For example, pembrolizumab is a PD-1 inhibitor that can treat skin melanoma, non-small-cell lung cancer, kidney cancer, bladder cancer, head and neck cancer, and Hodhkin lymphoma [12]. As another example, atezolizumab is a PD-L1 inhibitor that can treat bladder cancer, non-small-cell lung cancer, and Merkel cell carcinoma [12]. New inhibitors are being developed rapidly over time as more knowledge is being gained on the specifics of different cancers and their behavior. The examples mentioned in the forgoing are just a few under the broad umbrella of immunotherapy for which mAb plays an important role. The main objective of immunotherapy is to enable the immune system of patients to recognize or target specific cancer cells and destroy them [13]. In 2005 the total mAb therapeutics entering first-in-human studies per year is 35, 16 out of which are for cancer treatment. In 2017 the total mAb therapeutics rose to 105, and nearly 80 were for cancer treatment. Indeed, the antibody therapeutics entering clinical study and being approved are in record numbers [14].

Extracellular proteins secreted from yeasts are produced for making insulin, human serum albumin, and hepatitis vaccines. Insulin drug has reached over USD 24 billion market in 2018 according to a market study in 2019. The fast-growing biopharmaceutical business in producing therapeutic proteins is getting so popular that all major drug manufacturers also carry a parallel line of this business.

Unfortunately, the protein expressed from the bioprocess is in very small amounts in a large volume of suspension, that is, low concentration. The two key hurdles in recombinant DNA techniques to produce therapeutic protein [15] are (a) to recover this small concentration of protein after fermentation by separation and (2) to provide high purity of the protein product through separation and purification. It is prudent that both separation and purification processes should be robust and cost-effective for the biopharmaceutical technology to be viable and competitive. Although this text is focused on separation, one should bear in mind that given these two steps are sequential, poor separation can adversely affect purification downstream. Therefore it is prudent to have an integrated approach for downstream processing. To say the least, if there is an upset from the fermenter upstream producing, say, off-spec finer feed, the centrifuge should take on the upset feed and try to produce a consistent output downstream to the filter, membrane and chromatography column downstream in the interim, while the upset condition is being fixed. Otherwise, the entire chain of downstream processes can be seriously affected.

Other biotechnology involves synthesis and/or modification of intermediates or final products. Frequently, this is in a suspension form so that mechanical mixing, separation, spray or thermal...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• In God, I trust
• Preface to Second Edition (2019)
• Preface to First Edition (2007)
• 1. Introduction
• 2. Principles of Centrifugal Sedimentation
• 3. Batch and Semibatch Centrifuges
• 4. Disk Centrifuge
• 5. Decanter Centrifuge
• 6. Commercial Applications of Centrifugation in Biotechnology
• 7. Concentrating Solids by Centrifugation
• 8. Laboratory and Pilot Testing
• 9. Selection and Sizing of Centrifuges
• 10. Troubleshoot and Optimization
• 11. Visualization and Modeling of Flow and Separation in Tubular Centrifuge
• 12. Disk-Stack Modeling
• 13. Performance Projection of Centrifuges in Bioseparation
• 14. Rotating Membrane in Bioseparation
• 15. Flocculation With Decanter Centrifuges
• 16. Case Studies of Monotonic and Unimodal Size Distribution Models
• 17. Classifying Bimodal Particle Size Distribution and Case Study of Inclusion Body Classification
• 18. Integration of Unified Modeling With Practice in Centrifugal Separations
• Appendix A: Nomenclature
• Appendix B: Buckingham-π Analysis for Decanter and Tubular, Disk-Stack, and Spintube Centrifuges
• Appendix C: Centrate or Concentrate Discharge Through Rotating Impeller
• Appendix D: Answers to Problems in Chapters 2–17
• Index