BioTherapi: Bioinformatics for Therapeutic Peptides and Proteins

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Proteins are sensitive molecules held together by weak physical interactions and disulfide bonds. Their three-dimensional structure can be disrupted by a number of factors including the presence of hydrophobic surfaces, high shear, the removal of water and a change in temperature or pH.
Protein formulation technology is an integral part of drug development.A change in a protein's structure can not only negatively impact its therapeutic effect but can also trigger adverse immune reactions to the drug. Thermal stability of the ingredients is a good indicator of protein formulation stability.A different approach to increasing stability is to modify the protein formulation directly. For example, steric stabilization or modification of the zeta potential of liposomes will influence the overall stability. Often, a solution change (pH, salts, additives) will lead to a significant change in the zeta potential.

Protein Formulation Approach to improve the delivery/stability propeties of protein.

1. Protein engineering(site directed mutagenesis):
I. Allows amino acid substitutions at specific sites in a protein i.e. substituting a Met to a Leu will reduce likelihood of oxidation.
II. Strategic placement of cysteines to produce disulfides to increase Tm.

2. Pegylation: PEG is a non-toxic, hydrophilic, FDA approved, uncharged polymer,Increases in vivo half life (4-400X),decreases immunogenicity,increases protease resistance,increases solubility & stability and also reduces depot loss at injection sites

3. Microsphere encapulation: Process involves encapsulating protein or peptide drugs in small porous particles for protection from insults and for sustained release. Crommellin has applied hydrogel-based microspheres techniques. Despite an emphasis on non-injectable delivery technologies for biologics, particularly pulmonary, hydrogel-based techniques are likely to continue to dominate research as scientists seek to minimise injection frequency and boost patient compliance.
Two Types of Microsphere
1. Nonbiodegradable
a) Ceramic particles
b) Polyethylene co-vinyl acetate
c) Polymethacrylic acid/PEG
2. Biodegradable
a) Gelatin
b) Polylactic-Polyglycolic acid (PLGA): a biodegradable and biocompatible polymer, to create microspheres with mainly hydrophobic characteristics.Although it is difficult to avoid a burst release with this technique, a selection of polymers and production processes allows the release of proteins over two to three weeks.However, the use of organic solvents and the gradual drop of pH inside the microspheres may affect the integrity of the protein.

4. Crystalization: For years, small-molecule drugs have been delivered orally in a crystalline state, which protects the active ingredient from being destroyed by the stomach's temperature and pH.The crystal production technique does not involve x-ray diffraction, which could help keep down the cost of goods.
A crystallization technology developed by Altus, produces highly stable, potent proteins that can be delivered orally in a capsule. The crystals help the proteins survive the harsh pH environment in the stomach and are effective without systemic absorption. In addition, molecules can be cross-linked in a crystal lattice for better stability and control over where the drug is delivered in the body. Applications include the delivery of enzymes that exert a therapeutic effect in the stomach.
But according to Robert Gallotto, new crystal technologies can be used to manage protein purity without significantly reducing yields (which increases the cost of goods). And, crystallization can increase protein concentrations. "You can fit billions of particles of proteins in a 10-μm crystal. You don't have interaction going back and forth which thereby improves stability and reduces aggregation".

5. Hydrogels: are hydrophilic polymeric networks capable of imbibing large quantities of water. These can avoid organic solvents and pH drops do not occur during release.Protein release from hydrogels is controlled by bulk degradation of the material rather than by surface erosion in other systems.
These concerns are not new to protein therapeutics specialists. Soluble proteins lack chemical stability in the body: they have very short half-lives and may degrade upon storage by aggregation, oxidation, or deamidation mechanisms.
Of course, protein molecules can be stabilized and delivered in numerous ways and no single method will be appropriate for all applications. Several new formulation and delivery techniques have made strides along the development pathway.