Section I: Overview
Recombinant protein drugs, also known as recombinant DNA protein drugs, and genetically engineered protein drugs, refer to the use of recombinant DNA technology to optimize the modification of the gene encoding the protein of interest, and use a certain vector to introduce the target gene into an appropriate host cell. A biologically active protein preparation obtained by expressing a protein of interest and subjected to extraction and purification techniques for the treatment or prevention of human diseases.
First, the development of recombinant DNA technology and recombinant protein drugs
- The establishment of molecular biology theory
On April 25, 1953, James Watson and Francis Crick, working at the Cavendish Laboratory at Cambridge University, published a paper entitled "Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid" in the journal Nature and found double helix structure of DNA. At the same time, Nature published two papers by Rosalind Franklin and Maurice Wilkins, which proved the DNA structure model and jointly opened the era of molecular biology. Watson, Crick and Wilkins shared the 1962 Nobel Prize in Physiology and Medicine.
In 1958, at the 12th Symposium on Experimental Biology in the United Kingdom, Francis Crick presented the Central Dogma of Molecular Biology and published it in the proceedings of the seminar (Symp. Soc. Exp. Biol. 12).
In January and April 1965, Marshall Nirenberg published a paper, "RNA Codewords and Protein Synthesis" in Science and PNAS, deciphering the nucleotide genetic code, and thus achieved his collaboration with Har Khorana and Robert Holley in 1968 who got the Nobel Prize in Physiology and Medicine.
- Discovery of multiple tool enzymes
In 1956, Arthur Kornberg of the University of Washington isolated DNA polymerase and shared the 1959 Nobel Prize in Physiology and Medicine with Severo Ochoa.
In 1967, DNA ligase was discovered almost simultaneously in laboratories such as Gellert, Lehman, Richardson, and Hurwitz.
On July 28, 1970, Hamilton Othanel Smith of Johns Hopkins University published a paper "A Restriction enzyme from Hemophilus influenzae *1I. Purification and general properties" in the journal J. Molecular Biology, confirming restriction enzymes. And thus achieved the 1978 Nobel Prize in Physiology and Medicine with Werner Arber and Daniel Nathans.
- Establishment of DNA recombinant technology
On October 1, 1972, Paul Berg of Stanford University published a paper "Biochemical Method for Inserting New Genetic Information into DNA of Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda Phage Genes and the Galactose Operon of Escherichia coli" in PNAS. This indicates that the recombination of DNA molecules was completed in vitro and shared the 1980 Nobel Prize in Chemistry with Fredrick Sanger and Walter Gilbert.
On November 15, 1973, Stanley Norman Cohen of Stanford University and Herbert Boyer of the University of California, San Francisco collaborated in the publication of the paper "Construction of biologically functional bacterial plasmids in vitro" in the journal PNAS, which announcd the arrival of recombinant DNA technology and genetic engineering.
- Recombinant protein drugs come out
On August 24, 1978, David Goeddel of Genentech and others used E. coli to express and synthesize the world's first recombinant human insulin.
On May 14, 1982, Eli Lilly submitted a request for the listing of human insulin, the world's first recombinant protein drug, to the U.S. Food and Drug Administration (FDA). On October 28, 1982, the FDA approved the market for the drug (Humulin®). In 2017, the series still ranked the ninth in the global diabetes drug market with sales of US$1.34 billion/year.
Second, the expression system and basic procedures of recombinant protein drug preparation
The preparation of recombinant protein drugs mainly includes prokaryotic expression system and eukaryotic expression system. The prokaryotic expression system is mainly bacteria, and the most commonly used are Escherichia coli and Bacillus subtilis. The eukaryotic expression system is complex and has eukaryotic cell expression, including yeast cells, baculovirus-insect cells, mammalian cells, and transgenic animal and plant bioreactors.
Like most drug development processes, recombinant protein drug development mainly includes three parts: pharmaceutical research, pharmacology research and clinical research. However, unlike traditional small molecule drugs, even if the drug preparation process has been determined, the preparation process of the recombinant protein drug will largely determine the quality of the drug and directly affect the safety and effectiveness of the drug. Therefore, analysis and discussion from the development process of recombinant protein drugs will help us better understand the preparation process.
The pharmaceutical research of recombinant protein drugs mainly includes two stages: laboratory research and pilot research. The laboratory research is generally divided into the construction of recombinant engineering cells (including animal and plant bioreactors), cell culture and protein expression, and separation and purification of target proteins, protein pharmaceutical preparations, and quality research and quality control modules throughout. The pilot study mainly includes modules such as pilot process amplification, quality standard establishment and stability test. Pharmacological research mainly includes modules such as major pharmacodynamics, pharmacokinetics and safety evaluation.
When the target protein is isolated and purified to obtain a certain purity of the protein stock solution, after determining the preliminary formulation to prepare the pharmaceutical preparation, or after the laboratory basically determines the drug preparation process and produces at least one batch of samples that have passed the preliminary test (small test), it should be timely to carry out the stability study of protein and its preparations, the research data can be used as an important basis and content of the stability test; however, in the application of clinical materials, at least one batch of three consecutive trial samples must be completed under the pilot scale amplification process and the stability data of the sample should last for 6 months.
Samples prepared in laboratory tests can be used to carry out major pharmacodynamics and pharmacokinetic studies, while samples for safety evaluation studies in principle use samples should be prepared by pilot studies. This can better reflect the safety of actual production products and only samples prepared on a pilot scale can meet the sample size required for the test.
When drug research and development enters pharmaceutics pilot research and pharmacology research, it can be called into the preclinical research phase. In the laboratory pharmaceutical research stage, or only the initial pharmacodynamic or pharmacological test, it can not be called the preclinical research stage.
To be continued in Part Two…