Understanding the Basics of Peptides and Proteins

While both proteins and peptides possess numerous properties that offer significant therapeutic potential, there are fundamental differences between the two compounds. This article examines some similarities and differences between proteins and peptides in light of potential market applications, manufacturing techniques, and regulatory environment.

Definitions
Peptides are short polymers formed from the linking of (usually less than or equal to 100) amino acids, and comprise some of the basic components of human biological processes, including enzymes, hormones, and antibodies. The link between one amino acid residue and the next is known as a peptide bond or an amide bond – formed when a carboxyl group reacts with an amine group of an adjacent residue – giving the chemical its name.  
Proteins, by contrast, are typically much longer chains of (greater than 100) amino acids, similarly linked by peptide bonds. They play a critical role in biochemical reactions within cells.  Proteins are ubiquitous in cellular chemistry and structure and are crucial for carrying out most of the biological functions of living organisms.     
There are various conventions to determine the distinction between peptides and proteins, however generally speaking peptide chains are short and proteins long.  
Applications and Markets

Driving the therapeutic implementation of proteins and peptides is ‘The Human Genome Project’, which led to the initial sequencing of DNA to identify approximately 20,000-25,000 genes of the human genome from both a physical and functional standpoint.
Developments in manufacturing, including transgenic, recombinant and synthetic methods have been essential as protein and peptide drugs move into the mainstream. Peptide and protein therapeutics, especially antibody drugs, are attractive due to their high specificity and potency and low incidence of toxicity.

A recent report by market and technology research firm Frost & Sullivan indicated that more than 40 approved peptide-based drugs are in use today and approximately 800 in are being developed to treat allergies, cancer as well as Alzheimer's, Huntington’s, and Parkinson’s diseases (1).
The market for protein-based drugs is also promising. In a study released last October, BCC Research indicated that global market for protein therapeutics was worth $86.8 billion in 2007 and an estimated $95.2 billion in 2008. This is expected to reach $160.1 billion in 2013 for a compound annual growth rate (CAGR) of 10.9 percent.
A great deal of research is driven by the unique requirements of peptides and proteins, especially with regards to drug delivery systems.  Many life science companies are embracing new drug development approaches to proteins and peptides to provide formulations that are stable, have effective bioavailability, and enable sound manufacturing.
For example, parenteral, nasal and controlled release delivery technologies have evolved to deliver these compounds better.  Likewise, strides are being made in areas such as oral delivery, transdermal delivery, pulsatile, and on demand delivery of peptides and proteins.
Peptides typically offer low toxicity, high specificity, and demonstrate fewer toxicology issues compared to other small molecules drugs, and in many cases lead to the development of therapies that would be otherwise difficult to commercialize.
Protein drugs have received enormous attention from pharmaceutical companies due to their bioreactivity, specificity, safety and overall success rate. Yet, there are still improvements to be made, especially with respect to costly production and formulation and delivery methods. With advances in protein drug delivery, expansion of many drug markets and an increase in patient compliance is a high probability.  

Peptides Manufacturing Techniques
Peptides are manufactured utilizing two distinct techniques, solid phase and solution phase. Each has unique applications and their implementation can greatly affect the cost and scalability of the pharmaceuticals that incorporate their respective peptides.  
Liquid- or solution-based peptide synthesis is the older of the techniques, with most labs using solid-phase synthesis today.  The method is better for shorter peptide chains and is still useful in large-scale production greater than 100 kg in scale.  
Solid-phase synthesis allows for an innate mixing of natural peptides that are difficult to express in bacteria. It can incorporate amino acids that do not occur naturally and modify the peptide/protein backbone.  In this method, amino acids attach to polymer beads suspended in a solution to build peptides.  They remain attached to beads until cleaved by a reagent such as trifluoroacetic acid.  This immobilizes the peptide during the synthesis so it can be captured during filtration.  Liquid-phase reagents and by-products are simply flushed away. The benefits of solid-phase synthesis include higher speed of peptide production as it is a relatively simple process and easy scale-up. It is also more suitable than solution-phase synthesis for longer sequences.

Within solid-phase there exist two different methods, (t)ert-(B)ut(o)xy(c)arbonyl, or t-Boc, and 9H-(f)luoren-9-yl(m)eth(o)xy(c)arbonyl, or Fmoc.  
T-Boc is the original method used in solid-phase synthesis. It uses acidic condition to remove Boc from a growing peptide chain.  The method requires the use of small quantities of hydrofluoric acid, which is generally regarded as safe and specialized equipment.  This method is preferred for complex syntheses and when synthesizing non-natural peptides.
Fmoc was pioneered later than t-Boc and makes cleaving peptides uncomplicated.  It is also easier to hydrolyze the peptide from the resin with a weaker acid. This eliminates the need for specialized equipment.  Again, both methods are valuable and each suit specific applications.  However, Fmoc is more widely used because it eliminates the need for hydrofluoric acid.

Protein Manufacturing Techniques
Manufacturing biotech drugs is a complicated and time-consuming process, and it can take many years just to identify the therapeutic protein, determine its gene sequence and validate a process to make the molecules using biotechnology.
Prior to advances in biotechnology such as rDNA and Hybridoma cell technology, the few protein drugs available were derived from human and animal corpses. In fact, the human growth hormone was taken from human corpses, and the insulin required to treat diabetes was collected from slaughtered pigs. Given their source, these drugs were expensive and available in limited supply.

Hybridoma cell and rDNA technologies, however, have provided a cost effective way to produce protein-based drugs in bulk quantities.
Hybridomas are the fusion of tumor cells with certain white blood cells. This fusion causes endless replication for use in the production of specific protein-based drugs called monoclonal antibodies, which are effective in treating cancers and other ailments.
The introduction of rDNA technology, or genetic engineering, has allowed the gene that encodes for the required protein to be transferred from one organism into another, enabling larger amounts of the drug to be produced.
As part of the process, host cells that have been transformed to contain the gene of interest are grown in carefully controlled conditions in large stainless-steel tanks. The cells are then stimulated to produce the target proteins through very specific culture conditions, including maintaining a suitable balance of temperature, oxygen, and acidity among other variables. After careful culture, the proteins are isolated from the cultures and put through a rigorous test at every step of purification before being formulated into pharmaceutically active products.

This complex process is bound by the ‘Food and Drug Administration’s Sterile Drug Products Produced by Aseptic Processing-Current Good Manufacturing Practice’, which includes two central themes:
o    Ensure robust product protection through adequate design and control of equipment and facilities
o    Ensure that the operational and raw material inputs are predictable through adequate quality control and quality assurance.
 
The guidance has influenced industry to adopt better contamination prevention practices and a higher assurance of process consistency is expected to reduce the incidence of sterile drug manufacturing problems. This facilitates the ongoing availability of often therapeutically significant pharmaceuticals.
The steps involved make protein synthesis a more complex and costlier process as compared to peptide synthesis as it involves removing contaminants, such as viruses or bacteria, from the compound that could pose health risks.

Regulatory Implications
The manufacture of protein- and peptide-based drugs has really formed a symbiosis between the laboratory and the manufacturing environment.

Along with the guidance on aseptic processing, the development of these therapies is bound by other current Good Manufacturing Practices (cGMPs), specifically the risk-based approach to the development of protein- and peptide-based therapies and comparability protocols.
According to the FDA, the intensity of oversight necessary is related to several factors, including the degree of a manufacturer's product and process understanding and the robustness of the quality system controlling their process. 

For example, changes to such complex molecules as proteins and other naturally derived products that are made with complex manufacturing processes may need more regulatory oversight.  Moreover, process changes with critical variables that have not been sufficiently defined may require the submission of additional data or comparability protocols.  
In other cases, the FDA indicates that changes in well understood processes could be managed under a firm’s change control procedures.  Additional factors in performing risk-based quality assessments include instances when manufacturing processes are crucial to the safety of the product or when products serve a critical medical need or have a critical public health impact.
At the same time, FDA also applies risk-based principles to the product quality review process to aspects of investigational new drugs (INDs); pre-approval chemistry, manufacturing and controls (CMC) and post-approval supplement processes. 

Additionally, a comparability protocol mandated by FDA describes specific tests and studies, analytical procedures, and acceptance criteria to be achieved to demonstrate the lack of adverse effect for a specified type CMC change that may relate to the safety or effectiveness of the drug product.

Conclusion
The promise of peptides and proteins will not only reinvigorate drug innovation and discovery, it will also challenge the very ingenuity of pharmaceutical developers to develop novel delivery methods for present and future therapies.  The benefits of peptides and proteins in effectively treating disease and other life-threatening conditions outweigh per units costs.
Looking at the wide range of possibilities these compounds present, development of therapies and cures is sure to increase.  Knowledge of the methods of production, purification, and optimizing yield, a solution yield can result, maximizes the use of peptides and proteins in today's pharmaceutical research and development.

Sources:
Therapeutic Peptides in Europe: Finding the Opportunities, November 2004; Himanshu Parmar, Pharmaceutical-Biotechnology Analyst, Healthcare Practice; http://www.frost.com/prod/servlet/market-insight-top.pag?docid=28323554

Drug delivery systems and routes of administration of peptide and protein drugs Sanders LM., Syntex Research, Palo Alto, California 94303
Bulinski JC (1986). "Peptide antibodies: new tools for cell biology". Int. Rev. Cytol. 103: 281–302. PMID 2427468.