Introduction to Biologic and Biosimilar Product Development and Analysis

By Karen M. Nagel

The purpose of this book is to give a concise introduction to development and analysis of pharmaceutical biologics for those in the pharmaceutical industry who are switching focus from small molecules to biologics processing, analysis, and delivery.   In order to maintain a limited focus, Introduction to Biologic and Biosimilar Product Development and Analysis, will deal only with peptides, proteins and monoclonal antibodies.

 

• Language: English
• Publisher: Springer
• Release date: Sep 27, 2018
• ISBN: 9783319984285

Book preview

© American Association of Pharmaceutical Scientists 2018

Karen M. NagelIntroduction to Biologic and Biosimilar Product Development and AnalysisAAPS Introductions in the Pharmaceutical Scienceshttps://doi.org/10.1007/978-3-319-98428-5_1

1. Principles of Recombinant DNA Technology

Karen M. Nagel¹  

(1)

Chicago College of Pharmacy, Midwestern University, Downers Grove, IL, USA

Karen M. Nagel

Keywords

Recombinant DNA technologyGlycosylationPEGylationFusion proteinsBiopharmingNew Animal DrugProtein characterization

Introduction

Recombinant DNA technology and other aspects of biotechnology are a far newer area of pharmaceutical research and development than areas related to small molecule pharmaceuticals, and the methods employed in all areas of the drug development process, from drug discovery to the manufacturing protocols, equipment, control parameters and testing methodologies required by the FDA are substantially different than those used with small molecule drugs. Beginning with the elucidation of the structure of DNA, advances in molecular biology techniques have led to dramatic progress in medical research, disease diagnosis and drug development and have introduced a new vocabulary to the pharmaceutical industry.

General issues with discovery, production, purification, characterization and analysis of products that fall under the general heading of pharmaceutical biotechnology will be summarized in this chapter. The analytical methods covered here are frequently discussed in the primary literature, and a basic understanding of what the methods are will be useful when evaluating clinical trial literature, and will lay a foundation for the remaining chapters. Subsequent chapters will deal in more depth with specific issues related to protein and peptide pharmaceuticals, and monoclonal antibodies. It will also serve as the knowledge base for the final chapter, which is focused on regulatory issues, most notably, how they affect the approval of biosimilar products.

Martin states the basic idea in pharmaceutical biotechnology is to employ biological processes and biological molecules to create drugs and vaccines. Our ability to do just that has increased dramatically in the past several decades, as many areas of science and technology increase their knowledge base and are able to be successfully integrated with each other. Biochemistry, genetics, microbiology, molecular biology, engineering, and even computer technology combine with the more traditional pharmaceutical sciences disciplines of medicinal chemistry and pharmaceutics to improve the ability of researchers to develop therapies that would not have been possible even 30–40 years ago, either from a production standpoint, or a financial one. Now, an entire branch of the pharmaceutical industry is focused on developing biotechnology-derived products, and the large pharmaceutical corporations either possess their own biotechnology divisions, or contract with or purchase smaller firms that focus on these areas of research. Without the development of recombinant DNA technology, this would not have been possible [1].

Recombinant DNA technology is, put very simply, the capacity to edit DNA. The implications of this, given that DNA is the chemical basis for the hereditary properties of the cell, are broad. The rapid progress in this field can be better appreciated when considering that DNA was accepted as the genetic material scientists had been searching for less than 70 years ago. Prior to work by Hershey and Chase in 1952, DNA was believed to be too simple chemically to contain the genetic information needed for the development and functioning of living organisms. The now-familiar double helical structural model of DNA followed in the next year, and experiments on the material soon greatly expanded our knowledge of how gene expression is regulated in all organisms. The necessary tools for DNA manipulation rapidly developed at this time. Several of these tools will be discussed in greater length in the production section of this chapter. It is worth noting that while the methods described in this chapter are used routinely and safely now, when they were first introduced in the 1970s and 1980s, there was a great deal of public concern over the use of genetically engineered bacteria and fear over the perception that scientists were tampering with nature. While those concerns have been largely allayed, there are still related areas of science and research that meet with public opposition, most notably genetically modified food, and cloning technologies [2].

Production Methods

Cloning and Recombinant DNA Technology

In 1973, Herbert Boyer, Stanley Cohen and colleagues published the results of a collaborative research project in which they fused segments of frog DNA into a plasmid vector containing a gene for tetracycline resistance, forming a recombinant DNA (rDNA) molecule. The plasmids were then transferred into a strain of tetracycline-susceptible E. coli, plated on a growth medium containing tetracycline, and allowed to colonize. The colonies that incorporated the tetracycline resistance gene were able to grow. Some of the colonies were also found to have incorporated the ribosomal frog DNA [2, 3].

This research was an important beginning in the field of recombinant DNA technology, and subsequent studies went on to prove that the sequences of DNA that are required to code for a particular protein can be isolated, fused into a bacterial plasmid, replicated in a host cell such that the daughter cells contain the recombinant DNA molecule, and, assuming the DNA segment still contains appropriate signals for gene expression, encouraged to produce proteins encoded by the foreign DNA segment. In other words, there was potential to produce large amounts of protein without having to isolate it from its normal source. This had dramatic implications in health care, as a number of therapeutic proteins in use at the time were available in limited supply due to the scarcity of organs from which they could be isolated. Growth hormone obtained from cadaver pituitary glands is a key example of this. In other cases, proteins were isolated from animal products, and did not contain the identical amino acid sequence as the human protein, leading to some allergic responses in the recipients, even when the animal protein only differed from human by one or two amino acids. Bovine and porcine insulin were examples of the latter case [2–4].

The construction of an rDNA molecule occurs by first cutting DNA into smaller lengths using restriction endonucleases , enzymes that recognize specific sequences of base pairs and cut DNA at those specific points (Fig. 1.1). This allows the exact piece of DNA needed to express a target protein to be removed and isolated. The restriction endonuclease also allows the plasmid to be cut open at the same DNA sequence, opening the circle and making room for the foreign DNA to be spliced within [2–4].

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Fig. 1.1

Formation of a recombinant DNA molecule

The next step in the procedure requires another enzyme, DNA ligase . This enzyme anneals the sticky ends of the vector and foreign DNA, yielding a slightly larger circular unit, the recombinant molecule. In this situation, sticky does not have the normal definition, but instead refers to the fact that the bases on the so-called sticky ends form base pairs with the complementary bases on the other DNA molecule. It is important to note that DNA from different species can be combined in this way, for example, with animal DNA being combined with bacterial DNA. Because of this, rDNA is sometimes referred to as chimeric DNA, in reference to the chimera in Greek mythology, a monster composed of parts from a lion, a goat and a serpent [2–4].

An analogy involving motion picture film is worth considering when thinking about the above processes. In this case, individual frames of film are analogous to DNA nucleotides; a set of frames, or a movie scene, would encompass a gene that codes for a protein of interest. Scissors (or restriction endonucleases) could then be used to cut a scene from one movie and splicing tape (or DNA ligase) used to insert it into a completely different one [5, 6].

At this point, the DNA of interest needs to be produced in large quantities in order to eventually produce useful amounts of protein or be utilized for other important tasks such as detection of infectious disease causing agents or genetic mutations. Two procedures are used for this task: molecular cloning, and the polymerase chain reaction .

Molecular Cloning and Subsequent Protein Production by Fermentation Tank or Biopharming

Molecular cloning, often referred to as the Cohen-Boyer method , used the above process of creating an rDNA molecule, and then inserting the molecule into a cell for expression purposes, a step referred to as transformation. The transformed cell then replicated, with each daughter cell containing the DNA segment of interest. The method was initially slow, and even with modifications, was a relatively time-consuming and cumbersome process.

While bacterial cells were used initially and remain common, a wide variety of cell types may be utilized as host cells for the plasmid, including yeast cells, animal cells grown in culture, plant cells (such as tobacco or rice), and transgenic animals. Mammalian cell cytoplasm extract may also be used in order to express proteins in a cell-free system. All of the expression systems currently in use have advantages and disadvantages. If low cost and high yield is the main priority, bacterial and yeast systems are generally preferred. They are not ideal, however, if a larger protein is being expressed, as the cell size is a limitation. Additionally, if post-translational modifications such as glycosylation are necessary for proper protein function, bacterial cells are not appropriate and a yeast or mammalian cell line will be required. Glycosylation will be discussed in greater detail in a later section of this chapter [1, 4, 7].

Once the cell has been transformed, it needs to replicate. This is generally done by fermentation in a large industrial scale fermentation tank or bioreactor. Cell culture is a complicated process, and it is not a trivial procedure to convert a small scale laboratory culture procedure to large scale production. Depending on the cell type being used, a number of conditions must be optimized for proper cell growth. In general, cells containing the rDNA molecules are cultivated in large vessels containing a liquid growth medium that is optimized for pH, oxygen tension, temperature, and nutrient content [1, 8].

Bacterial and yeast cells are typically easier to grow than mammalian cells, but all require precise conditions for optimal growth. Many pharmaceutical proteins are grown by fermentation processes, most commonly involving E. coli or the yeast S. cerevisiae. If mammalian cells are used, the growth medium tends to be more complex, and may require sugars, amino acids, electrolytes, vitamins, growth factors, hormones, fetal calf serum, and other ingredients. Components, such as fetal calf serum, often contain contaminating proteins and complicate the purification steps that occur following production. They also have variable composition, due to differences in the animal from which the serum was obtained, time of year, supplier’s treatment of the serum, and infectious material to which the animal may have been exposed. Because of the potential problems related to serum use, including prion transmission if infected bovine, sheep or goat serum is used, serum-free formulations have been created by media suppliers, and have been used with satisfactory results in some cases [9, 10].

A growing area of research in protein production is that of biopharming , or growing proteins inside of plants or animals and then isolating the protein from the plant or animal. While this method may initially appear to be more cumbersome than simply growing the protein in a fermentation tank, a number of advantages do exist. Animal cells used in fermentation tanks are highly inefficient, and a process that requires ten thousand liters of cell culture media could potentially yield only 1–2 kg of useable protein. Developing a protein that could be expressed either in a plant, or in the milk of a common dairy animal could lead to large scale production of the protein in question by allowing the plant to grow and be harvested, or by milking the animal. The protein could then be extracted from the plant or milk. A number of proteins are being investigated in such systems (Table 1.1). At this time, only four have been approved by the US FDA (Table 1.2) [4, 11–14].

Table 1.1

Proteins being investigated for possible development in biopharming systems (partial list)