Laboratory Testing Blog

The recent increase in development of protein and peptide therapeutics demands highly informative methodologies.  More than 600 biological drugs are currently approved by FDA.  The interest in analyzing peptides and proteins arises from the need, during preclinical and clinical studies, to quantify levels of the metabolites in protein and peptide drugs.

Typical oligonucleotides used as therapeutics or potential therapeutic compounds are 15 to 30 nucleotides (nt) long.  Oligonucleotides <15 nt can be readily resolved by HPLC technology.  However, the separation of longer sequences are more challenging.  Ion-pair reversed-phase (IP-RP) HPLC has been traditionally used for oligonucleotides analysis.  The traditional IP-RPLC eluent system typically uses TEA or TEAA or HFIP and the ion-pairing agent triethylammonium ion and a C18, column at 60^oC.  Under such conditions, the HPLC column's hydrolytic stability becomes crucial and is important to prevent the potential contribution of the oligonucleotide secondary structure from impacting retention.  To overcome this, Waters has an ACQUITY UPLC BEH column that has demonstrated excellent performance for oligonucleotides of similar structures.

The efficient characterization of antibody drugs is important to both regulatory agencies and pharmaceutical companies to ensure the safety and efficacy of biopharmaceutical products.  Proteins with different sizes may show similar hydrophobicity and are therefore difficult to separate by traditional reversed-phase chromatography (RP).  

Generally, several methods are used to determine the pattern of disulfide bond linkages.  Among them, crystallography (R-Ray) and NMR are excellent tools that can identify disulfide bond linkages with minimal disulfide bond intermolecular exchange.  However, the application of both techniques is limited by large sample requirements, sample purity (>95%) and protein size.

The general approaches for full characterization of glycoproteins focus on two aspects: carbohydrates and the protein itself.  Methods that detects released carbohydrates are limited by the fact that it is almost impossible to achieve 100% release, therefore 85% is generally considered as an acceptable release extent.  

Monoclonal antibodies or mAb's, form the fastest growing segment of the pharmaceutical industry, with total annual sales expected to top $50 billion in the next four years.  23 full-size monoclonal antibodies and three monoclonal antibody fragments have been launched so far, several having quickly reached ‘blockbuster status’ with annual sales of over $1 billion.  Between 1995 and 2007, the number of monoclonal antibody-based drug candidates entering clinical trials more than tripled, and this expansion is continuing.

Protein and peptide drugs cover a broad range of clinical application, such as bacterial infections, inflammatory and rheumatic diseases, pain, hypertension, stroke, AIDS and cancer.

Developing new drugs, especially monoclonal antibody drugs can be a very complex and time-consuming process.  It's no wonder that the failure rate of these types of drugs is higher than conventional compounds.