EarlyPK for therapeutic peptide in rat serum

Pharmaceutical companies extensively utilize therapeutic peptides to surpass the limitations of therapeutic proteins. These peptides possess high specificity due to their small size and exhibit minimal toxicity, as evidenced by research. Moreover, therapeutic peptides are known to induce less immune response compared to recombinant proteins or whole antibodies.

 

Comparison between LC-SRM and RIA analytical methods

Pharmaceutical companies must conduct PK (pharmacokinetic) studies to assess the therapeutic properties and measure the degradation of their therapeutics. However, a significant concern in the utilization of therapeutic peptides lies in their metabolic instability, making it imperative to determine their half-life.

 

After the development of immuno-based assays, it is essential to validate them through cross-validation using complementary methods. Considering the specificity properties, employing the LC-SRM (Liquid Chromatography-Selected Reaction Monitoring) approach for the entire therapeutic peptide seems to be an interesting strategy.

Context of the PK study

• 54 rat serum samples from 6 different animals
• In order to assess the degradation kinetic, each animal was sampled at different times following drug injection.
• Results obtained were compared to RIA results.

 

Radioimmunoassay (RIA) is an analytic method used to measure the presence and concentration of specific drugs or their metabolites in biological samples, such as blood, urine, or saliva. The RIA method involves labelling one of the components, either the drug or the antibody, with a radioactive marker, which allows for the measurement of the binding reaction.

LC-SRM Analytical Strategy

Extracting therapeutic peptides or small proteins from complex matrices is a tough task. This is because certain interactions between proteins and peptides need to be disrupted within these matrices. The extraction of therapeutic peptides can be accomplished through various methods, depending on their molecular weight. In our case, the therapeutic peptide, ranging from 10 to 15 KDa (kilodalton), was successfully extracted from our serum sample using an optimized protein precipitation protocol.

 

Afterwards, a targeted analysis using LC-MS/MS (Liquid Chromatography-Tandem Mass Spectrometry) was carried out on a triple quadrupole mass spectrometer. In this analysis, the LC-SRM (Selected Reaction Monitoring) mode was employed.

 

LC-MS/MS analysis by LC-SRM is a technique used in analytical chemistry to identify and quantify specific molecules, such as proteins or peptides, in a complex sample. The process involves two main components: liquid chromatography (LC) and tandem mass spectrometry (MS/MS). In LC, the sample is separated into individual components based on their chemical properties using a liquid mobile phase and a stationary phase. This separation allows for better detection and analysis of the molecules of interest. Once the molecules are separated, they enter the mass spectrometer. In tandem mass spectrometry (MS/MS), the instrument performs several stages of mass analysis. The LC-SRM mode in LC-MS/MS analysis allows for the precise monitoring of selected precursor ions and their corresponding fragment ions. This targeted approach enhances the specificity and sensitivity of the analysis, enabling accurate quantification of the molecules of interest.

 

Conduct of the analysis:

1. The precursor ion, which corresponds to the entire therapeutic peptide to be analysed, is selected in the Q1 quadrupole.
2.  The selected precursor ion undergoes fragmentation by collision-induced dissociation in the Q2 collision cell. This process breaks the precursor ion into smaller fragments.
3. Specific fragment ions are selected in the Q3 quadrupole for detection and quantification. These fragment ions are characteristic of the target molecule and serve as unique signatures for its identification.

 

The combination of the precursor ion and its corresponding fragment ion is referred to as a “transition”. This transition is crucial as it provides both specificity and sensitivity for accurate quantification.

 

Figure 1 : Representative scheme of Selected reaction monitoring principle

Results

Figures 2 and 3 illustrate the comparison between LC-SRM results and RIA results for animals subjected to different initial doses (low or high).

 

Figure 2 : Kinetic of compound degradation for animals treated with low dose. Blue: RIA results for 3 animals. Orange: LC-SRM results for 3 animals.

Figure 3 : Kinetic of compound degradation for animals treated with high dose. Blue: RIA results for 3 animals. Orange: LC-SRM results for 3 animals.

The results indicated that the degradation of the compound could be effectively monitored using both RIA and LC-SRM methods. Regardless of the initial dose of the compound, comparable results were obtained using LC-SRM and RIA techniques. The cross validation of the findings is thus demonstrated in Figures 2 and 3.

However, 96 hours after sampling, for the animals treated with the high initial dose, the drug was not detected with SRM, while it remained detected with RIA (Figure 4).

Figure 4 : Compound dose decreases depending on time. Blue (RIA) results mean for 3 animals for high initial compound dose (triangle) or low initial dose (round); Orange: LC-SRM results mean for 3 animals for high initial compound dose (triangle) or low initial dose (round). 

Two hypotheses can be confronted:

• The first is that LC-SRM may be less sensitive than RIA for quantifying the compound of interest. 
• The second hypothesis suggests that RIA may measure not only the therapeutic peptide but also its metabolites, thus lacking specificity compared to LC-SRM.

 

In the case of a high initial compound dose, the results showed that RIA tended to provide higher quantitation of the compound at all-time points. As mentioned earlier, the difference could be attributed to the fact that RIA technique measures not only the therapeutic peptide but also its metabolites.

 

In this study, the LC-SRM approach was employed to validate the RIA PK study of a highly effective therapeutic peptide. The LC-SRM method proved to be a valuable approach for confirming and enhancing the results obtained through RIA analysis.

 

One of the key advantages of the LC-SRM approach is its ability to provide high specificity in distinguishing the therapeutic peptide from other naturally occurring peptides within the body, as well as its potential metabolites. This specificity is crucial for accurately quantifying and tracking the therapeutic peptide’s presence and concentration in biological samples.