Pharmaceutical Biotechnology

A Focus on Industrial Application

Adalberto Pessoa, Michele Vitolo, Paul Frederick Long

• Publisher: CRC Press
• Year: 2021
• ISBN:
  9781000399899
• Edition: 1
• Topic: Medicine
• Subtopic: Pharmacology

Pharmaceutical Biotechnology: A Focus on Industrial Application covers the development of new biopharmaceuticals as well as the improvement of those being produced. The main purpose is to provide background and concepts related to pharmaceutical biotechnology, together with an industrial perspective. This is a comprehensive text for undergraduates, graduates and academics in biochemistry, pharmacology and biopharmaceutics, as well as professionals working on the interdisciplinary field of pharmaceutical biotechnology. Written with educators in mind, this book provides teachers with background material to enhance their classes and offers students and other readers an easy-to-read text that examines the step-by-step stages of the development of new biopharmaceuticals.

Features:

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• Discusses specific points of great current relevance in relation to new processes as well as traditional processes

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• Addresses the main unitary operations used in the biopharmaceutical industry such as upstream and downstream

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• Includes chapters that allow a broad evaluation of the production process

Dr. Adalberto Pessoa Jr. is Full Professor at the School of Pharmaceutical Sciences of the University of São Paulo and Visiting Senior Professor at King's College London. He has experience in enzyme and fermentation technology and in the purification processes of biotechnological products such as liquid–liquid extraction, cross-flow filtration and chromatography of interest to the pharmaceutical and food industries.

Dr. Michele Vitolo is Full Professor at the School of Pharmaceutical Sciences of the University of São Paulo. He has experience in enzyme technology, in immobilization techniques (aiming the reuse of the biocatalyst) and in the operation of membrane reactors for obtaining biotechnological products of interest to the pharmaceutical, chemical and food industries.

Dr. Paul F. Long is Professor of Biotechnology at King's College London and Visiting International Research Professor at the University of São Paulo. He is a microbiologist by training and his research uses a combination of bioinformatics, laboratory and field studies to discover new medicines from nature, particularly from the marine environment.

1 Fundamentals of Biotechnology

Michele Vitolo

Universidade de São Paulo

Contents

1.1 Introduction

1.2 Biological Molecules

1.2.1 Introduction

1.2.2 Proteins

1.2.3 Nucleic Acids

1.2.4 Virus

1.3 Basic Technologies of the Biotechnological Processes

1.3.1 Monoclonal Antibody Technology

1.3.2 Bioprocessing Technology

1.3.3 Cell Culture Technology

1.3.4 Tissue Engineering Technology

1.3.5 Biosensors Technology

1.3.6 Genetic Engineering Technology

1.3.7 Protein Engineering Technology

1.3.8 Antisense RNA Technology

1.3.9 Chip DNA Technology

1.3.10 Biocomputing Technology

1.4 Biotechnology and Applications

1.4.1 Medical

1.4.1.1 Diagnostics

1.4.1.2 Therapeutics

1.4.2 Environment

1.4.3 Livestock Raise and Agriculture

1.5 Matters of Business

1.5.1 Structuring of a Biotechnology Enterprise

1.5.2 Biopharmaceutical Industry

1.6 Social Aspects of Biotechnology

1.6.1 Use of Bioproducts

1.6.2 Genetic Privacy and Laboratory Prognostic Testing

1.6.3 Stem Cells and Cloning

1.6.4 Use of Test Subjects

1.6.5 Agriculture

1.7 Final Considerations

References

1.1 Introduction

Biotechnology can be defined as an applied science that exploits biological systems (e.g. molecules, organelles, cells, tissues or whole animals or plants) to find solutions of economic value that benefit humankind. Within the context, we consider the very latest uses of biomolecules for therapeutic purposes, from antibiotics to more complex macromolecules and tissue systems defined as biological drugs. The definition of biological drug or biological product vary among regulatory agencies; they can also be known as ‘biologics’, ‘biological medicines’, ‘biological medicinal products’ or ‘biopharmaceuticals’. Most regulatory agencies, such as the American Food and Drug Administration (FDA) and the European Medicines Agency (EMA), consider biological products as a wide range of items including vaccines, blood and its components, allergenics, somatic cells, gene therapy, tissues and therapeutic proteins. As described by the FDA, ‘biologics can be composed of sugars, proteins, nucleic acids or complex combinations of these substances, or may be living entities such as cells and tissues’. In this book, the terms ‘biopharmaceutical’ and ‘biological drug’ are used interchangeably.

In spite of biotechnology becoming of immense scientific and industrial interest over the last three decades, biotechnological processes have been used by humans for millennia for beer, bread, cheese and wine production.

These food products were produced empirically without any theoretical knowledge about the processes involved. But, following the invention of the microscope by Antonie van Leeuwenhoek in 1667, the microbial world was discovered and could be studied. Knowledge about the characteristics of microorganisms (growth, metabolism, cell structure, reproduction, etc.) accumulated throughout subsequent centuries, culminating with Louis Pasteur’s (1876) conclusion that the fermentative process was a microbial process. As a consequence, fermentation technology carried out under no aseptic conditions grew so fast that several bioproducts such as ethanol, acetic acid, butanol and acetone quickly appeared on the market and in huge amounts.

Fermentations carried out under aseptic conditions and with pure microbial strains started around the 1940s and led to an explosion in the variety of new commercial bioproducts (antibiotics, steroids, amino acids, vaccines, enzymes, among others).

Pure microbial strains that could produce a specific bioproduct in high yield were obtained by the genetic improvement of wild strains. The main techniques employed were random mutations induced in the wild strains by exposure of the microbes to physical (UV radiation, for instance) and/or chemical agents or by mating between strains belonging to closely related species, followed by screening for more productive mutants using auxotrophic culture media. However, a great technical advance for modifying the metabolic characteristics of prokaryotic and eukaryotic cells occurred in 1972, when the techniques of cell fusion (hybridoma) and recombinant DNA were introduced. Both techniques enhanced the development of so-called genetic engineering, insofar as they allowed introducing specific and planned changes directly into cellular DNA. The era of empirical biotechnology was over.

1.2 Biological Molecules

1.2.1 Introduction

Living cells are composed of a large variety of macromolecules (labelled generically as biomolecules) such as carbohydrates (polymers of low MW sugars), proteins (polymers of amino acids), nucleic acids (polymers of nucleotides; DNA and RNA) and lipids (a heterogeneous group of compounds having high solubility in organic solvents in common).

Although carbohydrates and lipids are essential constituents of cells, from the viewpoint of biotechnology, proteins and nucleic acids are the main biomolecules of interest. Carbohydrates have a variety of roles inside the cell, such as energy storage, maintaining structure and stimulating the immune response (antigen–antibody interaction, amongst others). Lipids are also important energy stores within adipose tissue, but the most crucial role of lipids is as components of cell membranes, promoting fluidity of cell membranes as well as effector molecules in their own right such as PAF (Platelet Activator Factor). PAF is responsible for platelet aggregation during blood coagulation, for dilatation of blood vessels which mediates the inflammatory process and allergic response and for implantation of a zygote into the wall of the uterus.

1.2.2 Proteins

Proteins – from the Greek term meaning ‘first class’ – are unbranched polymers of amino acids joined head to tail, from a carboxyl group (called the C-terminus) to an amino group (called the N-terminus), through the formation of covalent peptide bonds, which are a type of amide linkage. Except for the amino acid glycine, all other amino acids have an asymmetrical C atom leading to enantiomers designed as L and D. Only L-amino acids are constituents of active proteins in living cells.

Peptide bonds are rigid and planar structures that favour folding of the primary structure of protein (the primary structure is the sequential order of amino acids in the chain). As a consequence, two main structural folds can appear along the protein chain, i.e., α-helix and β-pleated sheet. Often both types of folds are found in proteins intermixed with parts of the protein where the conformation doesn’t fit any regular pattern.

An α-helix can be visualized like a stick. It is stabilized by hydrogen bonds established between a peptide carbonyl group (C=O) of one amino acid and the peptide NH group of another amino acid, four residues farther up the chain. The α-helix can be oriented to the right (clockwise) or left (anti-clockwise), but the clockwise rotation is the most common pattern. Myoglobin is an example of a protein rich in α-helixes.

The β-pleated sheets resemble a concertinaed piece of paper sheet. It is stabilized by hydrogen bonds established between NH and C=O groups belonging to amino acids of adjacent peptide chains. The pleated sheet can exist in both parallel and antiparallel forms. In the parallel β-pleated sheet, adjacent chains run in the same direction (N → C or C → N). In the antiparallel β-pleated sheet, adjacent strands run in opposite directions. Ribonuclease is an example of protein rich in β-pleated sheets.

Regarding the architecture of protein molecules, it is by convention that we can distinguish four structural patterns: primary structure (corresponds to the sequence of amino acids along the chain and the position of disulphide bridges (S-S), if they exist); secondary structure (represented by α-helix and β-pleated sheets, both secondary structures stabilized by hydrogen bonds); tertiary structure (the polypeptide chains of protein molecules bend and fold in order to assume a more compact three-dimensional shape); quaternary structure (occurs when a protein is composed of two or more interacting polypeptide chains with characteristic tertiary structure, each polypeptide is commonly referred to as a subunit of the protein). It must be borne out that all the information necessary for a protein molecule to achieve its intricate architecture is contained within the amino acid sequence of its polypeptide chain(s). Whereas the primary structure of a protein is determined by the covalently linked amino acids in the polypeptide backbone, secondary and higher orders of structure are determined mainly by non-covalent forces such as hydrogen bonds, ionic bonds, and Van der Walls and hydrophobic interactions. In addition to the four structural levels cited, there are also the so-called super-secondary structures (which consist of aggregates of secondary structures) and domains (compact folding of amino acids – generally composed of 100–400 amino acids – found in a specific location of the protein molecule; the active site of an enzyme is an example of a catalytic domain).

In living cells, proteins have a wide variety of different functions: enzymes (e.g. catalase and trypsin), regulatory proteins (insulin, somatotropin), transport proteins (haemoglobin and serum albumin), storage proteins (ovalbumin, casein and ferritin), contractile and motile proteins (actin, myosin, tubulin), structural proteins (collagen, elastin, fibroin), protective proteins (immunoglobulins, fibrinogen and ricin) and exotic proteins (antifreeze proteins, monellin and glue proteins).

Simply described, the function of a protein is mediated by an interaction between the protein and a specific chemical activator (usually called a ligand) that results in a conformational change throughout the protein structure (Figure 1.1).

FIGURE 1.1 Sketch of a conformational change induced within a protein following interaction with a specific chemical activator called the ligand.

1.2.3 Nucleic Acids

Nucleic acids – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) – are involved in the flux of genetic information, the control of gene expression and the control of cell metabolism.

DNA consists of two polynucleotide strands wound together to a form a long, slender, helical molecule (DNA double helix). Each strand is formed by nucleotides linked 3′–5′ by phosphodiester bridges. The nucleotides, in turn, are composed of deoxyribose, phosphate group and nitrogenous base (guanine [G], adenine [A], thymine [T] or cytosine [C]). The nitrogenous bases of one strand are complementary to the bases of the other strand, i.e., A pairs T and G pairs C. The pairing between bases is stabilized by hydrogen bonds (a type of weak chemical bond widely found throughout the biological world). In the double helix, the pair of bases is located inside the helix whereas the phosphodiester bridges and deoxyribose are outside the helix. The structure of DNA is essentially the same in all living cells, differing only in the number and sequence of nitrogenous bases (A-C-G ≠ G-C-A). DNA has the capacity to self-replicate due to the action of a specific enzyme called DNA polymerase. It is estimated that human DNA is composed of about 3 billion nitrogenous bases (Felsenfeld and Groudinein, 2003).

The summation of all hereditary information stored within chromosomes is referred to as the genome. So it is the genome that orchestrates the development and life of an organism. A chromosome is not a simple structure of weaving of DNA but is a complex of DNA, proteins (histones) and other molecules (methyl, acetyl, etc.). This complex is called chromatin. Normally, chromatin is wrapped in aggregates of histones, so that it is possible to insert long DNA chains inside small organelles such as the nucleus of eukaryotic cells and mitochondria. Moreover, parts of chromatin can condense –; hiding long stretches of a DNA molecule – or can expand in order to expose parts of DNA sequence to be a transcript by RNA polymerase. The genome is a complex non-stop biochemical machine that constantly suffers intense and unspecific modifications, so even homozygote twins are not truly genetically identical but can present different gene copies. In this way, one twin can suffer from diabetes whereas the other twin is healthy, for instance.

Ribonucleic acid (RNA) is also a polymer of nucleotides differing from DNA in the sugar type (ribose) and one of the nitrogenous bases, i.e., uracil instead of thymine. RNA can be linear or cyclic as well as single or double stranded. There are several types of RNA (Figure 1.2), the best known being messenger (_mRNA), ribosomal (_rRNA) and transfer (_tRNA). Each type of RNA has a specific role on the flux of genetic information. This occurs in two sequential steps named, respectively, transcription (in which the _mRNA is synthesized by RNA polymerase that copies a part of the DNA strand taken as the template) and translation (the _mRNA links to the ribosome followed by _tRNA that brings amino acids to the surface of the ri...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Table of Contents
• Preface
• Editors
• Contributors
• Chapter 1 Fundamentals of Biotechnology
• Chapter 2 Thermodynamics Applied to Biomolecules
• Chapter 3 Expression Systems for the Production of Therapeutic Recombinant Proteins
• Chapter 4 Molecular Biology: Tools in Industrial Pharmaceutical Biotechnology
• Chapter 5 Molecular Biology Tools: Techniques and Enzymes
• Chapter 6 Bioinformatics Applied to the Development of Biomolecules of Pharmaceutical Interest
• Chapter 7 Bioprocesses: Microorganisms and Culture Media
• Chapter 8 Sterilization in Pharmaceutical Biotechnology
• Chapter 9 Kinetics of Cell Cultivation
• Chapter 10 Bioreactors: Modes of Operation
• Chapter 11 Agitation and Aeration: Oxygen Transfer and Cell Respiration
• Chapter 12 Mammalian Cell Culture Technology
• Chapter 13 Purification Process of Biomolecules
• Chapter 14 Lipopolysaccharides: Methods of Quantification and Removal from Biotechnological Products
• Chapter 15 Enzymes: The Catalytic Proteins
• Chapter 16 Enzymes as Drugs and Medicines
• Chapter 17 Aspects of the Immobilization Technique
• Chapter 18 Biomolecules in Analytical Methods
• Chapter 19 Nanotechnology and Biopharmaceuticals
• Chapter 20 Biosafety Applied in Pharmaceutical and Biotechnological Processes
• Chapter 21 Pharmaceutical Quality System for Biotechnology Products
• Chapter 22 Techno-Economic Evaluation of Biotechnological Processes and Pharmacoeconomic Analysis
• Chapter 23 Perspectives for Pharmaceutical Biotechnology
• Index