Therapeutic proteins are often more specific than other pharmacological agents, requiring lower doses to achieve the same clinical effects, and reducing side effects. Their specificity, however, means that drug pharmacokinetic and bioavailability data require the execution of measurements at extremely low analyte concentrations. Another complication comes from matrix effects in biological samples (plasma, serum, urine) where sugars or phospholipids, for example, can mask proteins during assay. Additionally, over-abundance of endogenous proteins themselves can simply out-dilute therapeutic proteins unless the target is selectively boosted.
Currently, ligand-binding assays are still the industry and regulatory body standard, but liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods are gaining popularity as more efficient assays for biopharmaceutical drug development. Although more expensive and complicated than traditional methods, these assays are flexible and highly sensitive to the low analyte concentrations under investigation.
In their extensive review of current methods, Van den Broek et al. (2013) discuss the seven critical factors or assay validation steps integral to the success of LC–MS/MS assays for therapeutic proteins.1 These are:
- internal standard selection
- protein purification
- enzymatic digestion
- signature peptide selection
- peptide purification
- liquid chromatographic separation
- mass spectrometric detection
For any assay to perform reliably and with sufficient sensitivity, internal standard selection is paramount. In LC–MS/MS assays, the standards are either protein or peptide. It is therefore important that the chosen standards react similarly to the analyte during sample preparation. This is especially true during the enzymatic digestion step in order to accurately estimate final therapeutic protein concentrations.
Protein purification must also be optimized. Ideally, sample preparation removes the interfering or over-abundant matrix elements mentioned previously, thus boosting analyte levels but without significant sample loss. Purification can also maximize analyte exposure to the cleavage enzyme. Optimizing this step improves enzyme access without inducing amino acid modifications that would interfere with final assay validity.
Digestion usually involves denaturation — unfolding the protein to a single strand — and reduction, or the breaking of disulfide bonds. Although digestion might not be required for proteins between 5 and 16 kDa, this enzymatic preparation can increase assay sensitivity by focusing on a tighter range of representative peptide signals. Digestion methods must also be reproducible and not affect analyte recovery. Following digestion, the authors propose immunoenrichment as a peptide purification step to boost analyte concentrations and improve assay performance.
Final steps include optimizing signature peptide selection by choosing reliable digestion products that are stable and perform well in assay. The authors conclude that the use of available software and databanks, such as PeptideAtlas, Skyline or Basic Local Alignment Search Tool (BLAST), is essential to thorough completion of this step.
This detailed and thorough review ends with discussion of current LC separation techniques and LC–MS/MS methodology. The authors note that using up-to-date technology can reduce lower limits of quantification (LLOQs) and streamline sample handling efficiency.
In comparison to immunoassay, the authors find that analyte recovery is greater and more sensitive using LC–MS/MS. They note that optimization of the new methods for each specific protein is challenging, suggesting that each of the seven critical steps need consideration.
In summary, the researchers prefer the development of appropriate and sensitive extraction/preparation procedures that focus on cleanup, selection and enrichment. Better digestion methods and automation of through-put indicate that LC–MS/MS shows great potential for biopharmaceutical therapeutic protein workflows in the near future.
1. van den Broek, I., Niessen, W.M.A., and van Dongen, W.D. (2013) “Bioanalytical LC–MS/MS of Protein-Based Biopharmaceuticals,” Journal of Chromatography B, 929 (pp. 161–79), doi: 10.1016/j.jchromb.2013.04.030.
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