By Gary Hu
American Peptide Company

With the strong growth in biologics, large molecules, and bio-pharmaceutical therapeutics in recent years, the pharmaceutical and biotech industries are increasingly turning toward peptides and proteins in the search for drug discovery targets. 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 environments.

Peptides are short polymers formed from the linking of 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 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 are 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 20,000 to 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 are being developed to treat allergies, cancer, and Alzheimer’s, Huntington’s, and Parkinson’s diseases.

The market for protein-based drugs is also promising. In a study released in October 2009, BCC Research indicated that the 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%.

A great deal of research is driven by the unique requirements of peptides and proteins, especially with regards to drug delivery systems. Many life sciences 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 delivery, and on-demand delivery of peptides and proteins. Peptides typically offer low toxicity and high specificity and demonstrate fewer toxicology issues compared with other small molecule 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 are high probabilities.

Peptide 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 byproducts 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 to 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 nonnatural 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 suits 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 sources, these drugs were expensive and available in limited supply.

Hybridoma cell and rDNA technologies, however, have provided cost-effective ways 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 FDA’s Sterile Drug Products Produced By Aseptic Processing — Current Good Manufacturing Practice, which includes two central themes:

  • ensure robust product protection through adequate design and control of equipment and facilities
  • 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 costly process as compared to peptide synthesis, as it involves removing contaminants that could pose health risks, such as viruses or bacteria, from the compound.

Regulatory Implications
The manufacture of protein- and peptide-based drugs has really led to a symbiotic relationship 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 its 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 in which 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, the FDA also applies risk-based principles to the product quality review process to aspects of investigational new drugs (INDs); preapproval chemistry, manufacturing, and controls (CMC); and postapproval supplement processes.

Additionally, a comparability protocol mandated by the 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 of CMC change that may relate to the safety or effectiveness of the drug product.

Reinvigorating Drug Innovation
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-unit 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 a solution’s yield can maximize the use of peptides and proteins in today’s pharmaceutical research and development.

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