Perspective Duggan, Vazvaei & Jenkins Key terms
Stable isotope-labeled IS: Compound that has the same chemical structure as the analyte, except that some of atoms of the molecule are replaced with the corresponding stable isotopes, such as, 13
C, 15 N, 2 H and 18 O. They can be
used in quantitative LC–MS/MS assays to normalize the response of an analyte for variations in sample preparation, injection volume, matrix effects and other instrumental conditions.
Digestion efficiency: In PrD-LCMS, the completeness or recovery of the digestion process. This is usually expressed as the molar equivalent ratio of surrogate peptide recovered from the final bioanalytical process to that of the nominal molar concentration of the biotherapeutic spiked into the quality control sample predigestion.
Monitoring peptide: Selected peptide that is unique to the target protein analyte and monitored to confirm or assess its structural integrity and evaluate assay robustness.
Stable-isotope-labeled peptide IS: Typically made by incorporating stable isotope containing amino acids into the surrogate peptide during chemical synthesis.
Stable isotope-labeled protein IS: Typically made by incorporating stable isotope containing amino acids into the protein during recombinant synthesis.
that might be applied prior to digestion. The use of sur- rogate peptide standards and different forms of stable isotope-labeled IS (SIL-IS) adds further consider- ations to these parameters. These stable isotope-labeled forms include: the unmodified stable labeled surrogate peptide, a ‘winged’ form of the stable labeled peptides IS with additional amino acid residues on the C- and N-termini, and stable labeled forms of the entire intact protein therapeutic (SIL-IS protein IS). In a typical recovery and digestion efficiency
experiment for the validation of a PrD-LCMS method with no immunoaffinity capture step using a simple surrogate peptide standard, three sets of QCs would prepared, each set at the low-, mid- and high-analyte levels as follows:
• Set A contains the analyte protein therapeutic spiked into matrix predigestion at L/M/H nominal levels, then digested and analyzed;
• Set B contains the analyte surrogate peptide spiked into matrix at concentrations equimolar to those of the analyte protein predigestion (L/M/H), then digested and analyzed;
• Set C contains extracted/digested/quenched blank matrix spiked with the analyte surrogate peptide at concentrations equimolar to those of the analyte protein postdigestion (L/M/H), then analyzed.
For each QC level the digestion efficiency is deter- mined by the average peak area of Set A divided by that
1392 Bioanalysis (2015) 7(11)
of Set B. The analogous ratio of Set B to Set C yields the recovery. More details regarding recovery and digestion
efficiency can be found in the White Paper [11]. As
is the case for bioanalytical methods, recovery of
small molecule LC–MS/MS the
surrogate
peptide and IS should be high enough to meet the method’s sensitivity requirements, and it should be consistent [7,8,9].
Protein assay stability considerations General stability considerations As
recommended by the regulatory guidances for
therapeutic molecules [8,9], the stability of protein biotherapeutics should be evaluated in solutions and biological matrices as necessary to produce valid and reliable data. The FDA and EMA guidance documents have well described the appropriate protein therapeutic stability experiments [8,9]. In this article, we will focus on special considerations for PrD- LCMS. One cau- tion regarding PrD-LCMS is that quantification of the protein therapeutic is achieved via a surrogate pep- tide, such that changes to other parts of the protein, not impacting the surrogate peptide, might be unde- tectable. Evaluation of any changes in monitoring peptides (peptides unique to the analyte but located in a different region of the molecule) may provide more confidence in the stability of the protein as a whole. Another important consideration is the possible pre- cipitation, aggregation or adsorption of analyte protein and peptide molecules. It is widely recognized that nonspecific binding of proteins and surrogate peptides to surfaces as well as protein aggregation or insolubility may appear as instability. Therefore, it is crucial that the method is appropriately tested and precautions applied rigorously to eliminate any potential factor that results in such biases that might appear as instability.
Stock solution stability If the reference standard is supplied in a solution form with a certificate of analysis/analytical report, the sta- bility of the solution is usually provided by the supplier. In contrast, if the reference standard is provided in pow- der form, or a different solvent system is used for the preparation of working solutions, then the stability of the stock solution (or working solution) should be deter- mined for a minimum of 6 h at room temperature [8,9]. Alternative storage temperatures and durations will need to be considered if there is a lack of stability under the conditions of use. For long-term storage (more than 1 week) it is recommended that the stock solutions be aliquotted in one-time-use portions to avoid mul- tiple freeze-and-thaw cycles and that long-term storage stability be established under these conditions.
future science group
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