Sub Topic | Secondary Topic: Analytical Methodology | HPLC (Biopharmaceutical Molecule)
Authors: Stefano Lanciotti, Patheon Inc. (Main Author); Daniele La Torre, Patheon Inc. (Presenting Author)
Presenting Author: Daniele La Torre
Purpose: The preliminary activities needed to support the GMP manufacturing of the CTM batch were performed using the HPLC system to test the Peptide and its purity profile. The native analytical method was claimed to be compatible with UPLC. However the chromatographic profile of the product obtained on a UPLC system showed a big hump eluting near the main peak. By means of selected investigational experiments it has been demonstrated that the anomalous chromatographic profile was attributable to the interaction of the Peptide with the stationary phase of the column and it can be related to the concentration/amount of the peptide in the sample injected.
Methods: The chromatographic analysis of the Peptide was performed by means of a UPLC system with UV detection at 220 nm. The elution gradient was made by using 0.02% (v/v) Trifluoroacetic acid and 1% (v/v) Acetonitrile in water as mobile phase A and 0.02% (v/v) Trifluoroacetic acid in Acetonitrile as mobile phase B. The separation was performed using a UPLC peptide 130 C18 column at a flow rate of 0.2 ml/min. The run time was of about 20 minutes. The reference standard and sample solutions, were prepared at nominal concentration of 1.0 mg/mL using as diluent solution water or Sodium Chloride 0.9 %. The separation was performed setting the column temperature at of both 40 °C and 60 °C. To support the investigation of the anomalous profile, the sample solutions were injected at increasing volumes (i.e. from 1µL up to 5µL) respect to the target value of 6µL. The UPLC method applied resulted in a chromatographic profile containing a big hump close to the main peak. The interest in performing a further investigation was mainly related to the fact that the above mentioned peak showed the same RRT of a product impurity. The hump is not present when analyzing the reference standard material. To this end, additional experiments were required to understand the root-cause of the anomalous profile. In details, the strategy was the following: study of the impact of the lyophilization process, analysis of final formulated solution before lyophilization, possible impact of the formulation process (e.g. pH adjustment, sequence of addition of excipients). In a second stage, it was also considered the impact of the chromatographic conditions applied during the analysis, with the concurrent evaluation of any sample aging-related phenomenon.
Results: The investigation started with the UPLC analysis of the formulated solution and the related lyophilized DP: both samples showed the hump. On the other hand a fresh made formulated solution without final pH adjustment did show a conforming profile. The above results indicate that the extra peak is not related to the lyophilization process of the product. Therefore we started the investigation on the formulation process starting from the pH adjustment. Two trials were performed using different concentrations of NaOH: samples were submitted to UPLC analysis. The results showed that the formation of the extra peak is visible in both samples. Then different DP solutions were prepared modifying the excipients addition sequence as to verify potential incompatibility of API with excipients. Results showed the extra peak in all formulations. The samples with the extra peak were submitted to Mass Spectroscopy characterization: data showed that the shoulder has the same pattern (mass/charge ratio) of the API peak, thus not supporting the hypothesis that this is a product related substance. Finally, the parameters of the analytical method were challenged together with the sample aging. Results showed that the extra peak is not related to samples aging. On the contrary the presence of the hump is strictly dependent on the sample injection.
Conclusion: The data obtained have demonstrated that the issue is not related to either the manufacturing process of the drug product or to sample aging but dependent on interaction of the Peptide with the stationary phase of the column and the concentration/amount of the sample injected for analysis.
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