Recombinant Protein Production

Evolving Paradigms of Recombinant Protein Production

• Limited natural availability of beneficial proteins restricts supply and use in therapeutics or industry
• Recombinant DNA (rDNA) technology uses microbes as surrogate hosts for protein production
• Microbial technology evolves and integrates modern innovations to improve recombinant biopharmaceutical production
• Strategies include fermentation, metabolic engineering, strong promoters, novel vector elements, protein tags, secretion signals, synthetic biology, cloning devices, and process screening
• Appraisal covers recombinant protein manufacture by microbes, biopharmaceutical production trends, and approaches for production
• Upstream process design (host/vector/promoter selection) is crucial
• Downstream process focuses on extraction and purification
• Review covers tools/methods for overcoming low expression and the cost of biopharmaceutical production and readily available protein products

Introduction

• Drug discovery and development are long, expensive processes
• Traditional treatments evolved from accumulated experience using natural resources
• Modern research shifted from small-molecule drugs to big macromolecules
• New medicine discovery starts by identifying a macromolecule, pathway, mechanism linked to a disease
• Human body produces proteins/enzymes for physiological functions like temperature, blood glucose regulation etc...
• Deficiency of biological proteins leads to pathological conditions like mental disorders, diabetes, impaired blood clotting
• Deficiency is treated with replacement proteins produced ex vivo in biological systems
• Therapeutic proteins must be highly pure, functional, and contaminant-free, and have a simple manufacturing process
• Choice of recombinant hosts, procedures, equipment, and production strategies must be planned for product quality
• Technology advancements lead to various recombinant products (peptides/proteins) on the market and in clinical trials
• Recombinant protein tech focuses on cell- or receptor-targeted proteins with reduced dose & side effects, increasing potency and targeting

Trends in Biopharmaceutical Proteins

• Biopharmaceuticals have transformed the pharmaceutical industry
• Biopharmaceutical proteins are a promising class of drugs due to specificity and effectiveness
• Complex nature requires precise characterization of protein structure and activity
• Article focuses on trends and emphasizes role of analytical technologies in development
• Explores evolving analytical tools for biopharmaceutical R&D
• Study of complex biotherapeutics and impurities requires advanced analysis for structure and function
• Mass spectrometry, NMR spectroscopy, electron microscopy, and X-ray crystallography are used for protein structure analysis
• Methods identify changes affecting effectiveness or toxicity
• X-ray crystallography reveals protein function and interactions
• Chromatography, capillary electrophoresis, and immunoassays are used for detecting and measuring impurities
• Microscopy reveals morphology, size, shape, and structure
• Sensitive analytical technologies ensure safety and efficacy

Recombinant Pharmaceutical Protein Production in Microbes

• Biotechnology began with successful expression of human insulin in Escherichia coli
• Humulin was approved by the FDA in 1982
• Market share is currently valued at $400 Billion
• Proteins are versatile macromolecules that make them precious for medicine
• Proteins play a role in genetic information as the major products and prime modulators of gene expression
• Recombinant DNA tech unlocked capabilities to produce existing and novel proteins through expression in hosts
• Manufacturing biomolecules using genetically engineered cells
• Any protein can be expressed if the gene sequence is known
• Humans have between 25,000 and 40,000 unique genes coded in their genomes [11]
• Recombinant protein production starts with cloning the target gene into a vector that would be:
  • Inserted into the expression host
  • Upstream bioprocessing
  • Downstream process to purify and formulate into end products
• Each phase has its own considerations to be optimized before embarking to ensure maximum efficiency in terms of costs, labor, and environmental

Microbial Host Selection

• Prokaryote and eukaryote hosts are explored for expressing human proteins: bacteria, yeasts, insect, mammalian cells, transgenic plants, and animals
• System considerations: manufacturing process and expression host
• Manufacturing: issues include bioprocess optimization, production capability/capacity, cost, regulatory requirements
• Host selection: considers gene-host compatibility, expressed protein properties, and inherent host characteristics
• Determining best host system for specific aims is critical
• Key differences between hosts: protein yield/productivity, post-translational modifications, and desired protein features
• Vectors and bacterial strains are main considerations for expression
• E. coli is most popular expression host, followed by yeasts (Saccharomyces cerevisiae and Pichia pastoris)
• Extensive research enables:
  • Fast dev of recombinant organisms
  • Efficient culture conditions
  • A simple downstream process
• Prokaryotic-efficient workhorses for non-glycolsylated proteins
• Eukaryotic-proteins that require post-translational modifications (PTMs) [20]

Escherichia coli

• Widely used to express recombinant products like therapeutic enzymes and proteins
• In 2012, 30% of therapeutic products from heterologous systems originated from E. coli and is likely higher now
• E. coli Benefits:
  • Superior growth kinetics
  • Accessible, cost-effective media requirements
  • Swift bacterial transformation
• E. coli genetics and biology are well understood, easing recombinant clone manipulation
• Challenges with E. coli: lacks PTMs, forms inclusion bodies (IB), low expression/inactive proteins from codon bias, and pyrogens
• Strains developed to overcome challenges: AD494 supports disulfide bond formation in cytoplasm; Rosetta addresses low expression of proteins
• Protein expression: secretory proteins, soluble proteins, and inclusion bodies (IBs) - intracellular protein aggregates insoluble and inactive
• Ideally, proteins should be secreted extracellularly or into the periplasmic space, but some strains deficient in secretory mechanisms and IBs are formed instead
• Proteins in IBs must be extracted, solubilized, and refolded, causing a 75-85% loss of recovered protein
• To fix IBs, fermentation can be set at lower temperatures and growth conditions manipulated by adding sugars and modifying pH

Yeasts

• Yeasts such as Saccharomyces cerevisiae, Pichia Pastoris, and Yarrowia lipolytica produce organic compounds, therapeutic (insulin) proteins, and interferons
• Yeast advantages:
  • non-pathogenic
  • easy to scale up
  • correctly folds proteins and performs PTMs
• Baker's yeast, S. cerevisiae, was first complete eukaryotic sequence published
• Genome understanding-valuable host for expressing recombinant proteins, genetic manipulations carried out with ease
• High expression levels extracellularly, which makes purification easier
• However, it causes intracellular accumulation that negatively impacts product yield and triggers cellular stress
• There is limited uptake of S. cerevisiae to produce therapeutic proteins
  • Unstable products
  • Reduced efficiency due to the hypermannosylation

Other Hosts

• Food-grade and widely accepted as safe, Lactococcus lactis is a bacterial host
  • perfect for generating recombinant proteins in the pharmaceutical industry for therapeutic applications
• Lactobacillus lactis Benefits and features: ability to secrete stable recombinant proteins into the growth medium with few proteases, resulting in properly folded, full-length protein, its gram-positive nature, preventing contaminating endotoxins, and its rapid growth to high cell densities, which facilitate large-scale fermentation
• Bacillus subtilis Benefits:
  • a high degree of genetic tractability, non-toxicity, economic growing medium, secretion capabilities, and a vast availability of genetic tools like promoter systems, shuttle vectors, and signal peptides [26,27]
• Additional yeasts for recombinant protein production include Hansenula polymorpha, Yarrowia lipolytica, Schizosaccharomyces pombe, and Kluyveromyces lactis, and non-conventional species
• New microorganisms are showing potential, like filamentous fungus Myceliophthora thermophila due to low viscosity during fermentation
• Myceliophthora thermophila facilitating its scalability
• Corynebacterium glutamicum is associated with recombinant protein production through constructs and secretion capabilities
• Microalgae hosts feature comparatively high growth rates and phototropic lifestyle, making them solar-powered and cost-effective
• Single-cell green algae (Chlamydomonas reinhardtii) and the diatom (Phaeodactylum tricornutum) exhibit secretion of antibodies

Vector and Promoter Systems

• Expression vector anatomy: replicon (origin of replication and control elements), promoter, operator, multiple cloning sites, and selection marker [17]
• Commercially available vectors include pET, pUC, and pQE series [22]
• Considerations of the vector: plasmid copy number and plasmid compatibility because they impact the organism and resulting protein yields
• Promoters are regulatory sequences that ensure efficient expression
• Promoters should:
  • Have capacity to ramp up production of proteins fast to 30%
  • Be controlled, using inhibitors or activators, to avoid toxicity
• Common promoter systems are:
  • Bacteria
    • lac, tac, and trc
  • Bacteriophages
    • T7, T5, and SP6 systems
• Promoters should be implanted into hosts simply, affected by culture media or inducers:
  • Strong
  • Simple and cost-effective
• LacI* gene is engineered into lacIQ to increase expression by 10-fold to deal with leaky expression

Upstream Process Development

• Includes cell line development, clone selection, and bioprocessing
• Fermentation requirements for E. coli are extensively documented, makes it easily bred on industrial scale
• Needs to confirm cells in bioprocessing will produce the quality and quantity of proteins
• Bioprocess development in small scale using test tubes and shaking flasks before bioreactor fermentation
• Key areas for Bioreactor Culture are optimization of media, fermentation system, and process parameters: influences transcription and translation of the cell
• Systems include tanks or single-use, must to be chosen between batch, fed batch, or continuous fermentation to ensure maximum cost/efficiency return
• To ensure process protein sustainable production commercially, parameters include pH, temperature, dissolved oxygen, carbon dioxide concentration, aeration, agitation of the slurry, and shear force
• Process intensification:
• address constraints and improve efficiency at a large scale
• involve managing feed, oxygen, cultivation, temperature, pH, and strain selection/ development

Downstream Process Development

• Purifying and obtaining the drug substance from a natural source
• Downstream process is obtaining "drug product"
  • Desired purity
  • Efficacy
  • Reasonable cost per unit
• Phase is critical to free candidate from any impurities, and must be sufficiently produced from the extraction
• Requires specific methods, techniques
• If Generated inside cells: crucial to extract and lyse cell followed by the removal of residual debris
• Several purification steps and analytical methods required ensure impurities are removed

Extraction

• The desired recombinant protein is obtained through the microbial cells
• Methods based on protein requirements:
  • centrifugation
  • sedimentation
  • flotation
  • microfiltration Extraction can be achieved by three different mechanisms:
  • Mechanical
  • Enzymatic

Mechanical Treatment

• Bead milling, heat shock, osmotic shock, impingement, high-pressure homogenization
• Physically break up the cell wall to release protein content
• Combines multiple processes such as slicing of the cell membrane using shear forces
• This method is rapid and may reduce risk of product contamination
• Also provides the ability to control temperature as the sample's released energy could affect the product

The Chemical Treatment

• Uses the usage of alkalis, organic solvents and detergents
• Used to recover molecular products
• Alkalai Benefit: Recover intracellular products if the target molecule that is expressed
• Comparably, solvents benefit: possess capacity to remove phospholipids
• Ionic detergents also benefit:
  • Denaturing membrane proteins to facilitate their solubilization and membrane extraction

Enzymatic Treatment

• Lysozyme, glucanase, mannose, proleases provide efficiency and selectivity
• Protein is sensitive and easily lost, requires reducing loss
• The enzyme's determination depends on the type of cell and composition Lysozyme works the best against gram-positive organisms over gram-negative accomplished by combining enzymatic activities of glucanases and proteases [44].

Purification

• Purpose is to ensure protein is free from an contaminants.
• Depends on protein properties optimization determines purity, yield, and functionality.
• Clarification occurs first: Remove insoluble cell debris, precipitates, and large particles.
• Subjecting the cell lysate to lower speed centrifugation helps remove debris and enhanced speed helps with removal of ribosomal materials
• Alternatives: ammonium Sulfate or polyethylene, Glycol fractionation, phase portioning, and membrane fillration techniques
• Chromatography is used to purify using affinity, Iin exchange, hydrophobic extraction, and size exclusion/Gel separation Important to get high purity proteins Reduce unwanted HCP production!

Product Concentration

• Final stage of downstream processes
• Concentrating the product & the formulation
• Removing all conts and misfolded parts
• HIC, SMB chromatography, crystallization, Refolding

Strategies to Overcome Low or No Expression

• High products come from high amounts of soluble proteins
• Several factors cause low production like...
• Overcome is possible!
• Medium composition plays a signifiant role, salts and yeast

Economics Of Microbial Products

• Biopharma: multi-dollar enterprise!
• Role in vaccine, therapeutics.
• Decreasing activity hurts economy...

Technologies In Those P

CRISPR: Improved potential for producing in bacteria.

Marketed Recombinant Microbial Products

• Recombinant: Completely new venture!
• Humulin

Conclusions

Advancements to promote protein!