Understanding the Basics of Peptide and Protein Production

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With strong growth in biologics, large molecules, and biopharmaceutical therapeutics in recent years, the pharmaceutical and biotech industries are increasingly turning toward peptides and proteins in their search for drug discovery targets. While both offer significant therapeutic potential, there are fundamental differences between the two types of molecule.

Definitions: Peptides are short polymers formed from the linking of (usually ≤100) amino acids. They comprise some of the most basic components of human biological processes, including enzymes and hormones. 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 this class of chemicals its name.

Proteins, by contrast, are longer chains of (>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 biological functions of living organisms. Scientists follow various conventions to determine the distinction between peptides and proteins. Generally speaking, however, 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 ~20,000–25,000 genes of the human genome from both a physical and functional standpoint. Developments in manufacturing — including transgenic, recombinant (rDNA), and synthetic methods — have been essential as protein and peptide drugs move into the mainstream. These molecules (especially antibodies) are attractive therapeutics because of their high specificity and potency and low incidence of toxicity.

A recent report by market and technology research firm Frost and Sullivan indicated that >40 approved peptide-based drugs are in use today, and ~800 are being developed to treat allergies and cancer as well as Alzheimer’s, Huntington’s, and Parkinson’s diseases (1). The market for protein-based drugs is also promising. BCC Research indicated in an October 2008 study that the global market for protein therapeutics was worth US$86.8 billion in 2007 and an estimated $95.2 billion in 2008 (2). This is expected to reach $160.1 billion in 2013 for a compound annual growth rate (CAGR) of 10.9%.

A great deal of protein/peptide research is driven by their unique requirements especially with regard to drug delivery. Many life science companies are embracing new approaches to provide formulations that are stable, have effective bioavailability, and enable sound manufacturing. Parenteral, nasal, and controlled-release delivery technologies have evolved to better deliver these medicines. Likewise, strides are being made in such areas as oral, transdermal, pulsatile, and on-demand delivery of peptides and proteins.

Peptides typically offer low toxicity and high specificity, and they demonstrate fewer toxicology issues than other small-molecule drugs. In many cases, those attributes lead to the development of therapies that would be otherwise difficult to commercialize. Protein drugs have received enormous attention from pharmaceutical companies because of their bioreactivity, specificity, safety, and overall success rate. Some improvements are yet to be made, especially with respect to costly production and formulation and delivery methods. Advances in protein drug delivery are expanding many drug markets and increasing patient compliance.

Manufacturing Techniques

Peptides are manufactured through three distinct techniques: solid phase synthesis, solution phase synthesis, and, and a combination of both. Each has unique applications, and their implementation can greatly affect the cost and scalability of pharmaceuticals that incorporate their respective peptides. Liquid- or solution-based peptide synthesis is the older method, but most laboratories use solid-phase synthesis (Figure 1) today. The former method is better for shorter peptide chains and still useful in large-scale production (>100 kg).

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 peptide/protein backbones. In this method, amino acids build peptides by attaching to polymer beads suspended in a solution. They remain attached to those beads until cleaved by a reagent such as trifluoroacetic acid, which immobilizes a peptide during synthesis so it can be captured by filtration. Liquid-phase reagents and by-products are simply flushed away. The benefits of solid-phase synthesis include faster production and easy scale-up because it is a relatively simple process. It is also more suitable to longer amino acid sequences than solution-phase synthesis.

There are two different solid-phase methods: tert-butoxycarbonyl (t-Boc) and 9H-f luoren-9-ylmethoxycarbonyl (Fmoc). T-Boc is the original method, which uses an acidic condition to remove Boc from a growing peptide chain. This requires using small quantities of hydrofluoric acid, which is generally regarded as safe for regulatory purposes, and specialized equipment. The T-Boc method is preferred for complex syntheses and when synthesizing unnatural peptides.

Fmoc (Figure 1) was pioneered later and makes cleaving peptides uncomplicated. It is also easier to hydrolyze a peptide from the Fmoc resin using a weaker acid, which eliminates the need for specialized equipment. 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. It can take many years just to identify a therapeutic protein, determine its gene sequence, and validate a biotechnology process to make it. Before advances such as rDNA and hybridoma cell technology, the few protein drugs available were derived from human and animal corpses. For example, human growth hormone (hGH) was taken from human corpses, and insulin for treating diabetes was collected from slaughtered pigs. Given their sources, such drugs were both expensive and limited in availability.

Hybridoma cells and rDNA technology have provided a cost-effective way to produce protein-based drugs in bulk quantities. Hybridomas are tumor cells fused with certain white blood cells. The fusion allows their endless replication for use in production of monoclonal antibody protein-based drugs, which are effective in treating cancers and other ailments.

Genetic engineering (rDNA technology) has allowed genes that encode for required proteins to be transferred from one organism or cell to another, enabling larger amounts of protein drugs to be produced. As part of this process, host cells transformed to contain the gene of interest are grown in carefully controlled conditions, traditionally in large stainless-steel tanks. The cells are then stimulated to produce those target proteins through very specific culture conditions, including maintaining a suitable balance of temperature, oxygen, and acidity among other variables. The proteins are isolated from those careful cultures and put through rigorous testing at every step of purification before being formulated into pharmaceutically active products.

Complex bioprocesses are bound by the US Food and Drug Administration’s current good manufacturing practice (CGMP) guidance on aseptic processing, which include two central themes (3): ensuring robust product protection through adequate design and control of equipment and facilities, and ensuring that the operational and raw material inputs are predictable through adequate quality control and quality assurance. Such guidance has influenced the biotechnology industry to improve contamination prevention practices. High assurance of process consistency is expected to reduce the incidence of sterile drug manufacturing problems, which facilitates the ongoing availability of often therapeutically significant pharmaceuticals. The steps involved in protein biological synthesis make up a more complex and costlier process than peptide synthesis because they involve removing contaminants (such as viruses or bacteria) that could pose health risks.

Regulatory Implications

Manufacturing protein- and peptide-based drugs has formed a symbiosis between laboratories and manufacturing plants. Along with the aseptic processing guidance, development of these therapies is bound by other CGMPs, among them a new risk-based approach to their development and comparability protocols (4,5).

According to the FDA, the intensity of necessary oversight is related to several factors, including the degree of a manufacturer’s product and process understanding and the robustness of the quality system controlling a process. For example, changes to such complex molecules as proteins and naturally derived products made by complex manufacturing processes may need more regulatory oversight. Moreover, process changes with critical variables that are insufficiently defined may require submission of additional data or comparability protocols. But the FDA indicates that changes in well-understood processes could be managed under a company’s own change-control procedures. Additional factors in performing risk-based quality assessments include cases in which manufacturing processes are crucial to product safety or when products either serve a critical medical need or have a critical public health impact.

At the same time, FDA is applying risk-based principles to its product quality review process for investigational new drugs (INDs); preapproval chemistry, manufacturing, and controls (CMC); and postapproval supplement processes. Additionally, an FDA-mandated comparability protocol describes specific tests and studies, analytical procedures, and acceptance criteria to demonstrate a lack of adverse effects for specified types of CMC changes that may relate to the safety or effectiveness of a drug product.

A Bright Future Ahead

The promise of peptides and proteins is not only reinvigorating drug innovation and discovery, it is also challenges the 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 their per-unit costs.

Looking at the broad range of possibilities these molecules present, development of therapies and cures based on them is sure to increase. Knowledge of the methods of production, purification, and optimizing yield will maximize the use of peptides and proteins in pharmaceutical research and development now and into the future.


1.) Parmar, H 2004. Therapeutic Peptides in Europe: Finding the Opportunities, Frost & Sullivan.

2.) Report BIO021C 2008. Protein Drugs: Global Markets and Manufacturing Technologies, BCC Research, Wellesley.

6.) Sanders, LM. 1990. Drug Delivery Systems and Routes of Administration of Peptide and Protein Drugs. Eur J. Drug Metab. Pharmacokinet. www.ncbi.nlm.nih.gov/pubmed/2200689 15:95-102.

7.) Bulinski, JC. 1986. Peptide Antibodies: New Tools for Cell Biology. Int. Rev. Cytol. PMID 2427468. 103:281-302.

8.) 2006. BioPortal BioBasics: Protein-Based Drugs, Government of Canada.

2 thoughts on “Understanding the Basics of Peptide and Protein Production

  1. Is there any evidence that proteins and peptides will help as a
    treatment for dementia lewy body?

    Would it or could it hurt the patient since lewy body patients seem to be quite sensitive to medications and supplemental?

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