Production of Recombinant Therapeutic Proteins
What are Recombinant therapeutic proteins?
Recombinant therapeutic proteins are human proteins engineered in the laboratory through recombinant DNA technology, which involves inserting the DNA encoding the proteins into a bacterial or mammalian cell or any other expression system, expressing the protein in these systems and then purifying the protein from them.
These proteins permit an individualized treatment approach by supporting a therapeutically targeted process and thus are important factors to compensate the deficiency of an essential protein.
Therapeutic proteins include hormones, cytokines, growth factors, and blood products like albumin, thrombolytics, fibrinolytics, antibiotics, vaccines, clotting factors (vii, vii, and ix), tissue plasminogen activator and many more
Significance of therapeutic proteins
Since the approval of insulin in 1982 more than 120 recombinant proteins have been approved and become available as extremely valuable therapeutic options. Over the past two decades the demand for therapeutic proteins is continuously increasing because of the following features of these proteins.
- Recombinant therapeutic proteins play their role in various life threatening diseases including end stage renal disease(erythropoietin), various infectious diseases, viral hepatitis(interferons), cancer and related diseases, clotting disorders(factor vii, viii, ix), diabetes(insulin),and inborn metabolism disorders(lysosomal enzymes).
- These recombinant proteins replace chemically synthesized drugs and are highly specific in their function
- They show less interference with normal biological process and are less likely to elicit immune responses
Production of various therapeutic proteins across the Globe
Classifying recombinant therpeutic proteins
Recombinant therapeutic proteins are classified into following major categories:
1) Therapeutic proteins with enzymatic or regulator activity
- Replacing a protein that is deficient or abnormal
E .g. somatotrophin, antithrombin III, lactase, pancreatic enzymes, insulin, human albumin, factor viii, factor ix,and many more
- Augmenting an existing pathway
For example erythropoietin, FSH, various interferons, urokinase, factor viia, salon calcitonin, trypsin and some others are also included.
- Providing a novel function or activity
For example botulinum toxin type, collagenase, papain, L-asparaginase, streptokinase etc
2) Therapeutic proteins with special targeting activity
- Interfering with a molecule of organism
Cancer and immunoregulation proteins, proteins given during transplantation, pulmonary disorders, infectious diseases, homeostasis and thrombosis, endocrine disorders and some other metabolic abnormalities
- Delivering other compounds or proteins
For example Denileukin diftitox (89, 90), ibritumomab tiuxetan88 etc
3) Therapeutic vaccines
- Protecting against a deleterious foreign agent
For example hepatitis B surface antigen, HPV vaccine, etc
- Treating an autoimmune disease
For example Anti rhesus, immunoglobulin G
- Treating cancer ( these proteins are currently in trials)
4) Therapeutic diagnostics
For example glucagon (288,289), growth hormone releasing hormone, TSH, HIV antigens , Hepatitis C antigens and many more
Percentages of various recombinant therapeutic proteins produced
Procedure for production of recombinant therapeutic proteins
The basic procedure for the production of recombinant therapeutic proteins involves:
1) Isolation of gene of interest with the help of restriction enzymes
2) Insertion of isolated gene to expression vector with the help of ligase
3) Transfer of recombinant vector to host cell
4) Identification and isolation of cells containing recombinant vector
5) Choosing an expression system for the production of recombinant therapeutic protein in host cells
6) Isolation and purification of proteins
Step 1:Isolating the gene of interest
Due to the development in recombinant DNA technology, in late1970’s, it became possible for the researchers to isolate any desired gene from any organism from which isolating intact DNA (or RNA) was possible. Bacterial genes were the first genes to be isolated and picked up by bacteriophage. By the isolation of the hybrid bacteriophages, the DNA for the bacterial gene was recovered in a highly enriched form. This formed the basic principal behind recombinant DNA technology.
Using Recombinant DNA technology investigators isolate a gene that is as little as 1 millionth part of the total genetic material present in an organism. The gene of interest is divided into a number of small pieces that are then placed into individual cells (usually bacterial). These cells are then separated as individual colonies on petri plates, and are screened through rapidly to locate the gene of interest.
The enzymes used to cut gene of interest as well as the vector, Restriction endo-nucleases, usually cut at defined sequences of (usually) 4 or 6 bp. This allows proper cutting of gene of interest at specific locations. Self-complementary (or "sticky") ends are generated at a 5’ overhang or a 3’ overhang by the restriction endonucleases due to an off-centre cleavage in the pseudo-palindromes, the recognition sites for endonucleases.
The sticky ends being complementary, can anneal to each other. Regardless of their origin (animal, plant, fungal, bacterial) any two fragments can join together and form recombinant molecules
Step 2:Insertion of isolated gene to a vector
A self-replicating DNA molecule which is used to transfer isolated genes/DNA segments between host cells is called a vector. It is usually a small molecule and has a single site for restriction endonuclease activity ideally.
Vectors are used to transfer DNA between species so they must meet three requirements:
- They must autonomously replicate in the host cell. The common vectors used in rDNA technology are designed for replicating in bacteria or yeast, but vectors for plants, animals and other species are also present.
- To distinguish the cells containing the recombinant DNA from those that do not, these vectors must contain a selectable marker .For example vectors for drug resistance in bacteria.
- An insertion site to accommodate foreign DNA is a must in vectors. This site is a unique restriction cleavage site in a non essential region of the vector. Nowadays, more than one restriction cleavage site are also found in a vector.
Vectors are classified into the following types.
Circular, Extra-chromosomal, Double-stranded, molecule of DNA in bacteria is called plasmid. It is the most common type of vector used in rDNA technology.
Using bacteria, millions of copies of the recombinant plasmid can be produced within an hour inside the bacteria and then this amplified vector can be extracted for further processing or manipulation.
ii) VIRAL VECTORS
Genetically engineered viruses which carry modified viral DNA or RNA that is noninfectious, but the presence of viral promoters and transgene in them helps to translate the transgene through a viral promoter. However, for large-scale transfection they require helper viruses or packaging lines, because viral vectors frequently lack infectious sequences,
Another purpose of these viral vectors is to incorporate the Gene of Interest into the host genome, and thus to leave distinct genetic markers in the host genome after incorporation of the transgene. For example, characteristic retroviral integration pattern of retroviruses after insertion of GOI is detectable and indicates incorporation of the viral vector into host genome.
A type of hybrid plasmid, the cosmid, contains a Lambda phage sequence (cos sequence). DNA sequences are originally from the lambda phage. Genetic engineering uses cosmids as cloning vectors. Genomic libraries can be built using cosmids
iv) BACTERIAL ARTIFICIAL CHROMOSOME
BAC is used for transforming and cloning in bacteria, usually E. coli, is a DNA construct, based on a functional plasmid (or F-plasmid),
Step 3:Transfer of recombinant vector to the host cell
There are several methods developed to introduce the recombinant DNA molecule into the desired host cell. These methods are adopted according to the type of vector used and the host cell into which this recombinant vector is to be introduced. Some of these are discussed below briefly.
Transformation is the Introduction of rDNA molecules into a living cell. The DNA molecule is made to come in the contact of cell surface. Then the host cell takes up this DNA molecule.
For transformation temperature shock of either high temperature (37- 45°C) or low temperature (0-5°C) is required. As reported by Mandel and Higa (1970) E. coli cells uptake DNA when treated with ice cold CaCl2 and exposed to 42°C for about 90 seconds.
The transfer of foreign DNA into cultured host cells mediated through chemicals is Transfection.
The DNA molecules are mixed with charged chemical substances such as calcium phosphate of DEAE dextran, cationic liposomes etc. Mixture of these chemicals is overlaid on the recipient host cells. Consequently the host cells take up the foreign DNA.
- Electric Field-mediated Membrane Permeation :
In electroporation a solution containing foreign DNA and fragile host cells, is passed through an electric current at high voltage (about 350 V). Due to this current, transient microscopic pores are developed in the cell membrane of naked protoplasts of host cells.
Consequently foreign DNA enters into the host protoplast through these transient pores. The transformed protoplasts are then cultured in vitro. This results in regeneration of the respective cell walls.
A glass micropipette with a very fine tip of about 0.5 mm diameter is used in this technique to forcibly inject foreign DNA into the nucleus host cells.
5. Gene gun/Particle Bombardment Gun:
In this method DNA is coated on macroscopic gold or tungsten particles. These particles are contained in a plastic micro-carrier and placed near rupture disc.
The bombardment apparatus then bombards them on the target cells. Thus the DNA is delivered into the desired host cells forcibly.
6. Agrobacterium-mediated Gene Transfer:
In this technique Agrobacteriumis used to transfer foreign DNA to host cells through its Ti-Plasmid. This recombinant plasmid is then inserted into A. tumifaciens cells. The cultured plant cell is then infected with genetically modified A. tumifaciens which delivers foreign DNA containing recombinant plasmid into the host plant cell.
Step 4: Identification and isolation of cells containing Recombinant vector
There are different methods to identify and isolate recombinant colonies. These include blue-white screening, colony PCR, positive selection vector, diagnostic restriction digest etc. but the most authentic and accurate way is by sequencing. In this method we isolate Plasmid DNA from an overnight bacterial culture. The insert is then identified by Sanger sequencing by using the sequencing primers suitable for the vector. To verify the exact sequence of insert it is required that Sequencing across the entire insert is done.
Step 5: Expression system for the production of recombinant therapeutic protein
Although the basic steps involved in rDNA technology for the production of recombinant therapeutic proteins is same, different expression systems are used for the production of various kinds of these therapeutic proteins. This is due to the reason that each kind of protein undergoes different modifications which are possible in specific type of expression system easily. Currently the expression systems used commonly are filamentous fungi, yeast, E.coli, mammalian cells, some plants and even insects.
The host cells first used for the production of therapeutic proteins, as an expression system, were bacterial cells especially E.coli. It was selected due to its simple physiology, short generation time and large yield of products. It worked well for insulin and growth hormones but soon it was realized that it lacked the ability to modify the proteins through glycosylation. Another drawback was that the synthesized protein if toxic to bacteria prevented reaching high densities of the required protein.
The yeast Saccharomyces cerevisiae is able to express some of the modifications of human proteins (e.g. N-glycosylation) and thus opted as an established host for the expression of certain types of therapeutic proteins like the human insulin glucagon, platelet derived growth factor etc. but as this glycosylation is so different from mammalian glycosylation process for many other types of proteins, concerns developed regarding severe immune responses in reaction to such an artificial glycosylation.
Mammalian cell lines
The most reliable host for expression of recombinant therapeutic proteins is mammalian cells. Natural glycosylation is provided by three mammalian cell lines derived from Cho, BHK and human fibrosarcoma cells. Another advantage of using mammalian host cells is the secretion of recombinant proteins in a natural form in media instead of in the form of the inclusion bodies (containing denatured proteins) in the cells in bacteria and yeasts. Other than these cell lines, additional production platforms are now being used as a trial and will be used commercially in near future.
Plants are also being metabolically engineered to be used as expression platforms but they have competitive cost than other systems.
Insects can also be used as expression systems for many types of therapeutic proteins because of the properties of high level of expression and correct folding of these proteins but the insects are expensive and difficult to handle. They have slow generation time and are not suitable for proteins with repetitive sequences.
None of the therapeutic proteins can be produced in all types of expression. The choice of expression system changes on Case-to-Case basis even for the same protein, because the general advantages of each expression system change in different situations and applications. But choosing an expression system distant from human race is theoretically the best option from safety point of view as human pathogens cannot attack those systems
step 6:Isolation and purification of proteins
Various methods can be adopted to isolate and purify the protein product from the host cells. These include centrifugation, electrophoresis and liquid chromatography.
It purifies particles on the basis difference in their mass or density.
- Differential centrifugation: it purifies soluble proteins from other insoluble materials of the host cell. The supernatant contains a mixture of all types of soluble proteins.
- Rate zonal centrifugation: separates proteins on the basis of their masses in a density gradient. All proteins separate into eh form of bands or discs starting from a thin zone at the top.
Purification of proteins on the basis of their charge to mass ratio is done through electrophoresis.
- SDS-PAGE: separation of proteins is solely on the basis of their charge to mass ratio in the form of bands which can be visualized using dye. Smaller proteins are at the bottom while larger proteins remain at top. It cannot differentiate proteins with similar masses.
- Two dimensional electrophoresis: To purify a mixture of proteins with similar masses, a two step process is used in which first step is to separate proteins on the basis of their charge difference (iso-electric focusing). And then in the next step SDS-PAGE is followed to separate the proteins on the basis of charge to mass ratio.
c) Liquid Chromatography
Purification of a protein mixture is done on the basis of difference in mass, charge or binding affinity. In this technique the sample of proteins is placed in a column of specialized beads contained in a glass or plastic cylinder. The nature of beads in the cylinder determines the type of liquid chromatography.
- Gel filtration chromatography: porous beads made from polyacrylamide, dextran (a bacterial polysaccharide), or agarose (a seaweed derivative), are used to separate proteins on the basis of difference in their mass. Larger proteins flow easily through the column and are eluted first while smaller proteins are held in the depressions of beads and are eluted late
- Ion exchange chromatography: specially modified beads whose surfaces are covered by carboxyl groups /amino groups and thus carry either a negative charge (COO) / positive charge (NH3) at neutral pH are used to separate proteins on the basis of difference in their charges. The proteins having charge similar to the beads are eluted first because of the repulsion. The other proteins are then eluted by adding a salt of high concentration.
- Affinity chromatography: the beads used to form the column are specialized by covalently attaching ligand molecules that bind to the protein of interest. Enzyme substrates or other small molecules that bind to specific proteins can be the ligands attached to the beads. Only those proteins that bind the ligand attached to the beads will be retained in the affinity column, the remaining proteins, regardless of their charges or masses, will pass through the column without binding to it.
Western Blotting A powerful method for detecting a particular protein in a complex mixture combines the specificity of antibodies, resolving power of gel electrophoresis, and the sensitivity of enzyme assays is called Western Blotting, or immune-blotting, this multistep procedure is commonly used to separate proteins and then identify a specific protein of interest.
Some therapeutic proteins approved for human use
Steps in production of recombinant therapoeutic proteins
Production of insulin
Insulin, a protein hormone, is composed of 2 different amino acid chains: "alpha" & "beta", which are held together by two disulfide bonds. There are 21 amino acids in "Alpha" chain and 30 amino acids in "B" chain, each of them arranged in a unique ordered sequence.
For the production of insulin, genes for the two insulin "A" and "B" chain were synthesized by the Scientists in the laboratory. Small pieces of DNA sequence were chemically linked together and then joined in a specific manner to form complete genes for the two chains.
To perform the molecular surgery using special enzymes, the genes synthesized were stitched into bacterial plasmids. These recombinant plasmids containing the transplanted genetic material for insulin were introduced into E. coli bacteria.
Inside the bacteria, translation of the genes for the production of Alpha and Beta chain proteins started after the genes were "switched-on" by the bacteria. This process of translation is identical to the natural translation of bacterial genes. When enough amounts of the "A" and "B" chains proteins were produced, bacteria were harvested to isolate these proteins and purify them. To form the complete Insulin molecule, the two chains were combined chemically in the laboratory identical to process in the human body.
Production of Insulin through rDNA technology
Manufacturing human insulin
Production of Hepatitis B vaccine
Human hepatitis B virus vaccine was prepared in yeast (Saccharomyces cerevisiae) using antigen produced by recombinant dna technology. Correct amino acid sequence was present in the highly purified antigen and this sequence assumed the appropriate conformational structure needed to trigger an effective immune response. As demonstrated in tests carried out in animals and in human beings, Yeast-derived vaccine is safe and is equally immunogenic and protective against hepatitis B as plasma-derived vaccine. On July 23, 1986 the yeast-derived vaccine produced by the Merck Sharp & Dohme Research Laboratories was licensed for general use in Germany and America
Production of hepatitis B vaccine through rDNA technology
Manufacturing Hepatitis B vaccine
- Recombinant therapeutic protein production in cultivated mammalian cells: Current status and future
Official Full-Text Publication: Recombinant therapeutic protein production in cultivated mammalian cells: Current status and future prospects on ResearchGate, the professional network for scientists.
- Recombinant therapeutic proteins
keywords: Recombinant therapeutic proteins,classification of protein therapeutics, Drug production, Recombinant proteins for human use, Transgenic animlas, Hepatitis vaccine, Follicle stimulating hormone, Cancer vaccine
- Recombinant therapeutic protein production in cultivated mammalian cells: current status and future
Keywords: Therapeutic proteins, cultivated mammalian cells, biopharmaceuticals, improved protein yields This article explains about current status of using mammalian cells as expression systems for therapeutic proteins and their future prospects
- Recombinant therapeutic proteins: Production platforms and challenges - Dingermann - 2007 - Biotechn
Keywords: Biotechnologicals; Glycosylation;Post expression modification;Recombinant proteins;Safety This article explains the production platforms used for manufacturing various therapeutic proteins and challenges faced in using these platforms
- Production of recombinant therapeutic proteins in human cells: current achievements and future persp
Protein Pept Lett. 2013 Dec;20(12):1373-81. Research Support, Non-U.S. Gov't; Review
© 2016 Sehrish Goher Ali