(M1330-08-47) Optimization of HPLC Method for Impurities Determination of Chromophore Molecule to Solve an Anomalous Carry Over Issue

Purpose: During the ICH shelf life stability analysis of Drug Product lyophilized vials (formulated as 25 mg per vial), for a commercial batch on the market, an unexpected and unknown peak was observed in the chromatographic profile. This API of this Drug Product is a chromophore molecule belonging to the tricarbocyanine group. This molecule is used as fluorescent tracer for medical diagnostics. A validated HPLC method is used for the quantification of impurities for release and stability purpose. After a deep evaluation of stability trend, an anomalous carry over was hypothesized, that generated an increase of the unknown peak during the sequence without impacting the main peak. In particular, after different trial injections, an increase of the only involved peak was detected (picture 1), but, strangely, on diluent solution this carry over was not visible (even if acquired after several sample injections) This issue obviously caused an overestimation of unknown impurities for sample injections (SST criteria applied to main peak did not allow to intercept the issue).

Methods: Instrumental settings of the HPLC method are summarized in picture 2: The indicated column ODS (5μm, 250 x 4.0 mm) was correctly used.

Results: Considering the serious impact on the batch present on the market, representative of the entire annual manufacturing, different investigative trials were executed in a very short time to understand the reason of this anomalous carry over and optimize the method. The investigation was managed with the aim to understand and solve the issue. Firstly it was verified the cleaning procedure of needle and HPLC injector, the HPLC configuration, challenging different HPLC models, detectors, brand and pipeline used without find out the clear root cause. In addition also the different HPLC vials configuration were challenged (amber and clear vial, stoppers pre-cut and not) without solving the issue. During this stage the column cleaning was challenged using different solvents (methanol and water are the best solvents for the column cleaning of tricarbocyanine molecule). The column cleaning was performed with different timeframe (increased hours up to overnight); the chromatographic profile showed good result in terms of impurity, but after few sample injections the issue reappeared, confirming that the impurity carry over was not due to a poor preliminary cleaning. The laboratory investigation allowed to circumscribe the nature of the event to the HPLC column performance and to exclude an issue linked to the /equipment/sample matrix/sample handling/cleaning/consumables. The attention was focused on the column performance and after a brainstorming with the column supplier, new investigative trials were necessary to challenge different HPLC columns; new ODS columns freshly activated (with same stationary phase batch or different stationary phase batch) showed the same carry over after few injections. Considering all the investigation performed and the outcomes obtained a scientific and deeply evaluation of the stationary phase was executed. It was found that the ODS column is an old configuration of the HPLC column, manufactured with a Silica type A. The Silica type A is a material with a high presence of metals inside. Metals that could be easily chelated by the tricarbocyanine molecule. An equivalent column was identified with the supplier: ODS‑3 column 250mm x 4.6 mm, particle size 5 µm. This column presents a new generation of Silica (3-series) with high purity and low amount of impurity in terms of contaminant and metals. Therefore, to scientifically confirm this hypothesis, a typical analytical sequence was run and the anomalous carry over issue on unknown impurity was finally solved, allowing the laboratory to certify correctly the stability data and continue the monitoring of the impurity level of the batch along the product shelf life. With this “new generation” column the results are very consistent, only a slight shift in retention time was noted, due to the different silica nature, in particular ODS-3 column has a relatively strong retentiveness compared to the old one, but this parameter does not influence the System Suitability criteria. A comparison of the two chromatographic profile in picture 3.

Conclusion: In light of this analytical deep investigation, the HPLC method was implemented, without affecting the original method validation in GMP area for a commercial product (USP chapter < 621 >). The new column generation, labelled with the same USP code (L1) of the older one, has been introduced and different preventive actions (cleaning and conditioning procedure for the HPLC columns) has been implemented in order to extend the shelf life of the column. After one year and several HPLC sequences run, it can be concluded that, with this simple implementation, no other issues of anomalous carry over has been obtained.

References: USP Chapter 621

Picture1 Chromatographic profile of the injections, with details of the observed carryover (from 0.2% to2.1% of unknown impurities)



Picture 2 Instrumental setting of HPLC method



Picture 3 Column comparison: old generation (ODS) on left, new generation (ODS-3) on right