Therapeutic recombinant proteins serve as a powerful tool to combat many diseases that were previously hard to treat. Chinese hamster ovary (CHO) cells and Escherichia coli are the most widely utilized expression systems. However, all expression systems have unique weaknesses and strengths concerning development cost, timeline, protein size, yield, growth conditions, regulatory approval, and post-translational modifications.
Efficient strategies for recombinant protein production services are becoming increasingly important as more applications requiring large quantities of proteins hit the market. High production efficiency and lower costs for the final product are required to ensure a commercially viable process. This article examines the challenges and opportunities in recombinant protein expression.
Challenges
Environmental Contamination
One of the biggest challenges affecting plant protein expression systems and solid phase peptide synthesis involves concerns surrounding genetically modified (GM) crops. There is significant concern about recombinant genes spreading through pollen dispersal, seed dispersal, and horizontal or viral transfer. There is also a risk of therapeutic proteins finding their way into the human or animal food supply, which could have an adverse effect on environmental organisms. In the past, the USDA legislated in response to incidents where transgenic plants were spotted in food crops. The use of ELISA kits to test for exposure to viruses and other pathogens has has identified instances of environmental contamination.
ELISA tests utilize specialized enzymes attached to antibodies to detect specific antigens.
Several strategies could mitigate such concerns, including geographic containment and planting during different seasons than those used for local food crops. Other essential strategies include:
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The utilization of male sterility in strains of GM plants through chloroplast expression systems.
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The use of inducible promoters.
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The production of easily identified plant varieties, like white tomatoes.
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The use of self-pollinating species.
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The production of non-germinating seeds.
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The production of inactive fusion proteins that are activated by post-purification processing.
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Growing crops inside appropriately managed greenhouses.
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The use of hydroponic growing rooms or cell suspension cultures provides an economical and effective means of containing GM plant materials.
Loss of Expression
Efficient expression of the gene of interest is necessary for the optimal production of recombinant proteins in custom gene synthesis. The challenge is that expression can be lost due to structural changes in recombinant genes or the loss of genes from host cells. Let us examine the loss of expression across three alternatives: plasmids, viral delivery, or integration into the host’s chromosomes.
In Plasmid-based Systems
What are plasmids? They are extrachromosomal, self-replicating cytoplasmic DNA elements found in eukaryotes and prokaryotes. Plasmids are primarily used as molecular vehicles for recombinant genes in antibody sequencing services. The ease of manipulating plasmids makes them a popular choice for genetic expression. The gene dosage depends on the plasmid copy number and is higher than when integrating recombinant genes into the host’s chromosome. The plasmid copy number is highly dependent on the host, the plasmid, and culture conditions.
Notably, copy-control genes regulate the plasmid count, which ranges from just a few to over 200. Plasmids usually impose a metabolic load on the host because cellular resources must be utilized to replicate and express plasmid-encoded genes during antibody production. The increase in metabolic load is directly proportional to increases in temperature, insert size, expression levels, recombinant protein yield, and the level of toxicity of the expressed protein to the host.
Such metabolic loads commonly lead to a decrease in the growth rate of plasmid-bearing cells. As the metabolic load increases with the copy number, the growth rate decreases, leading to faster-growing, plasmid-free cells overtaking the culture. The leading cause of reduced productivity in recombinant proteins is plasmid loss. Unequal plasmid distribution during cell division eventually leads to plasmid-free cells, a phenomenon known as plasmid segregation instability.
Chromosomal integration
In gene synthesis, the chromosomal integration of the gene of interest serves as a powerful alternative to overcome the expression instability of plasmid-based systems. The host does not bear the plasmid replication and maintenance burden. Chromosome integration is mainly suitable for the metabolic engineering of the host. However, the adequate integration of a foreign gene into the chromosome is both time-intensive and labor-intensive. Additionally, chromosomal integration leads to lower production rates than plasmid-based systems because of a lower gene copy number.
Chromosome integration has been the preferred strategy for custom protein synthesis in the commercial expression of recombinant proteins using animal cells. In this case, the investment in the long and intricate procedure of developing the host is easily compensated for by having a stable host. The major problem with chromosomal integration is the possibility of integrating the gene of interest into an inactive region of the chromatin. The use of locus control regions is one of the main strategies to overcome this problem.
Viral vectors
One of the most effective and simplest ways to deliver a gene of interest for peptide synthesis is through viral vectors. Viruses have evolved to deliver their genetic material to a host in an efficient and non-destructive manner. Retroviruses are among the viral vectors that promote the integration of the viral genome into the cell’s chromosomes. Others are primarily used for transient expression, where the steps for producing recombinant proteins only take place during specific stages of the virus's life cycle.
The simplicity of virus-driven expression makes it essential for production in higher eukaryotes, as establishing stable recombinant animal cells is a long and tedious procedure. More often than not, this expression is utilized to generate sufficient proteins for polyclonal immune responses, preliminary drug candidate testing, or laboratory-scale applications.
Post-Translational Processing
Aggregation, Folding, and Solubility
What is protein folding in the context of gene synthesis services? Protein folding is a complex process in which two kinds of molecules play an important role: chaperones, which prevent the formation of non-native, insoluble folding intermediates, and foldases, which accelerate protein folding. Occasionally, folding does not proceed adequately, resulting in misfolded proteins accumulating in intracellular aggregates called inclusion bodies.
Cell stress is one of the major causes of incorrect protein folding, often caused by nutrient depletion, heat shock, and other stimuli. As a response to stress, cells increase the expression of various chaperones, specifically those from the families of Hsp70 and Hsp100. When inactive proteins are produced, it represents a metabolic load and an energetic drain on the host.
On the other hand, the accumulation of inclusion bodies leads to structural strain in cells. Incorrect folding often leads to adverse consequences, including several human pathologies like Parkinson’s, Alzheimer’s, and Huntington’s disease. These are characterized by intracellular protein accumulation and aggregation. Protein aggregation has often been observed not only in E. coli but also in yeast, bacteria, insects, and mammalian cells.
Proteolytic Processing
In solid-phase peptide synthesis and recombinant production, signal peptides required to direct proteins to various cellular compartments must be cleaved to produce a functional protein.
The signal peptide is removed by a membrane-bound signal peptidase complex upon translocation across the cellular membrane in prokaryotes or the endoplasmic reticulum in eukaryotes. Inefficient signal peptide removal results in protein retention and aggregation within incorrect compartments, such as the endoplasmic reticulum. Furthermore, the yield of secreted proteins can be drastically reduced.
Transport and Localization
As pointed out above, recombinant proteins can be directed to different cellular compartments through fusion proteins or signal peptides. Different protein localization sites bear distinct advantages and disadvantages. Intracellular accumulation typically leads to high yields of protein while allowing for the easy recovery of concentrated proteins within cells. However, the protein purification technique required for extracts from protein-rich cells is often quite tricky.
Opportunities
The market potential for therapeutic proteins is enormous, with products ranging from enzymes to vaccines and from antibodies to hormones. Peptide synthesis companies must overcome hurdles to ensure antibody specificity, as every type of recombinant protein presents unique production challenges. These challenges often align with the strengths of the different available expression systems.
It takes a relatively shorter time to go from sequence to the production of grams of protein through high-yield transient expression systems like magnification. This is a primary advantage that plants have over other expression systems. This strength lends itself to producing vaccines to treat emerging or rapidly mutating diseases, such as bioterror threats or influenza. Additionally, there is potential for small production runs through this technology for orphan diseases with few patients or personalized treatments. The combination of rapid output and the ability to grow transgenic plants in low-cost greenhouses could significantly reduce the otherwise high cost of protein drugs for rare diseases.
With the maturation of the therapeutic market, patents will expire, allowing for the production of ‘biosimilars’—which are original copies of licensed proteins produced off-patent. Plant expression systems, such as high-yield chloroplast expression systems, allow the larger-scale production of proven drugs at a lower cost, grown in fields or greenhouses with appropriate containment strategies in place.
With the current state of glycoengineering in plants, therapeutic proteins do not necessarily need human-like N-glycosylation because it is not yet naturally found in plants. However, with progress in engineering, in vitro glycosylation procedures, and glycosylation pathways, the production of N-glycosylated therapeutic proteins in plants may become possible in the future.
One major setback to the widespread acceptance of plant expression systems is the lack of regulatory approval. However, there are recombinant protein products produced from plants that are primarily veterinary, diagnostic, or classed as medical devices, which do not need to meet the high standards required for human drug use. Currently, the cost and difficulty of gaining this approval often outweigh the benefits of utilizing plants to produce therapeutic proteins.
The low cost alongside high scalability is one of the primary benefits of plant expression systems. While it is true that plants can produce protein more cheaply than mammalian cell cultures, their impact on the overall cost of producing therapeutic protein drugs is currently limited. The cost plays a more significant role in product purification, which is essentially the same whether using cell extracts from mammalian or plant cells. If protein purification and harvesting could be conducted at a lower cost in plants—perhaps through targeting expression to specific storage bodies—there would be a much more significant economic benefit.
Plants are also considered safer in comparison to other expression systems, as they do not produce endotoxins or naturally support the growth of viruses or prions that have the potential for human infection.
Growth Control
The growth rate affects many parameters that determine the accumulation rate of recombinant proteins. These include the percentage of substrates utilized for RNA polymerase activity, cellular maintenance, plasmid stability, ribosome counts, plasmid copy numbers, cell distribution in cell-cycle phases, and plasmid multimerization.
Therefore, it is possible to control recombinant protein production by modulating the growth rate. The growth rate is primarily manipulated through the availability of nutrients. The principal nitrogen and carbon sources can be maintained at predetermined concentrations to achieve the desired growth rate. Such manipulation is typically achieved via continuous or fed-batch cultures. Dissolved oxygen is another essential nutrient for aerobic cells and can also be utilized to control the growth rate. Finally, molecular biology approaches can be used to genetically manipulate the growth rate for optimal yields.
Bioreactor and Operation Strategies
Besides containment, a bioreactor's main objective is to control the environmental parameters at predetermined values. The complexity of the bioreactor determines the number of parameters that can be manipulated. This range can span from simple temperature control in static culture flasks to the manipulation of many parameters in a fully instrumented vessel.
The conditions that can be manipulated include temperature, dissolved oxygen, pH, agitation rate, redox potential, dissolved carbon dioxide, power input, volume, medium composition, pressure, substrate concentration, cell concentration, and cell growth rate.
Final Thoughts
The contemporary issues arising with the expression of proteins in mammalian cells are now more easily solved through cell engineering. For instance, when engaging in large-scale production, most cells suffer from metabolic pressures like oxygen depletion and the accumulation of toxic metabolites, which affect final yields.
The problems faced in recombinant protein production must be solved because recombinant proteins are essential to the development of the current biologics landscape.