Sample preparation

Standardization & Calibration

Chromatography

Measurement & Analysis

This report describes the application of reversed phase HPLC-ELSD (high performance liquid chromatography - evaporative light scattering detection) for the quantification of polysorbate 80 in an Erbitux^® antibody drug formulation (Erbitux^® is the trade name of the drug formulation using the monoclonal antibody cetuximab). Sample preparation is performed using solid phase extraction and a set of seven calibration solutions is prepared for system calibration.

EXPERIMENTAL PROCEDURE

The samples were received as Erbitux^® drug product (DP, formulation of 5 mg/mL cetuximab, excipients: sodium chloride, glycine, polysorbate 80, citric acid monohydrate, sodium hydroxide, water) and were stored at 8 °C. Prior to sample preparation the samples were heated up to room temperature. A set of two Erbitux^® DP batches was analyzed in this work. The polysorbate 80 concentration of the samples was approximately 0.1 mg/mL. No dilution was required before loading onto the solid phase extraction (SPE) cartridge. All solvents applied during sample preparation were of gradient grade HPLC quality or higher.

SAMPLE PREPARATION

The blank sample was represented by pure water and does not undergo the solid phase extraction process. The Erbitux^® DP samples were purified by SPE. In detail, sample preparation was executed as follows:

1. 4M Guanidinium hydrochloride solution
   Dilute 80 mL guanidinium hydrochloride solution 6M with 40 mL water.
2. 10% Methanol
   Dilute 10 mL methanol with 90 mL water to obtain a solution of 10% methanol in water.
3. Sample preparation – Solid phase extraction
   1. Position Supel^™ Swift HLB SPE cartridge in Visiprep^™ SPE Vacuum Manifold.
   2. Prime with 1 mL methanol.
   3. Condition with 1 mL water.
   4. Load 0.5 mL of an Erbitux^® DP sample solution, add 0.5 mL water.
   5. Wash with 1 mL 4M guanidinium hydrochloride solution.
   6. Wash with 1 mL 10% methanol.
   7. Elute with 1 mL acetonitrile and collect eluent in 15 mL centrifuge tube.
   8. Repeat elution once and collect eluent in the same centrifuge tube.
   9. Evaporate acetonitrile in vacuum rotary evaporator at 40 °C for 25 minutes.
   10. Reconstitute sample with 250 µL water, vortex mix well and then transfer to HPLC glass vials.

STANDARD PREPARATION

The preparation of calibration standards was performed as follows:

1. PS 80 standard stock solution 1.2 mg/mL
   For the preparation of PS 80 stock solution (c = 1.2 mg/mL) weigh approximately 60 mg PS 80 into a 50 mL volumetric flask and fill up to mark with water.
2. PS 80 calibration standards
   Prepare dilution series according to Table 1 to obtain a set of seven calibration standards.

RP-HPLC-ELSD SYSTEM SETUP

The essential settings of the Hitachi Chromaster chromatography system and the gradient conditions applied in the analysis of polysorbate 80 are listed in Tables 2 and 3 below.

DATA ANALYSIS

Data was processed with Chromeleon^™ 7.2.10 software; due to application of a gradient profile and the necessity to summarize peak areas in the retention time range from approximately 6 to 9 minutes, the integration was executed manually. Integration of peaks outside the mentioned range was inhibited automatically. The calibration type applied was “Quad with offset”.

CALIBRATION DATA

A total of seven polysorbate 80 calibration standards was prepared. Quadratic regression revealed an excellent fit of the resulting calibration curve over the entire calibration range, with an R2 value of 0.9997 (see Figure 2). Experimental data obtained from the calibration experiments are listed in Table 4. Figure 3 displays an overlay of the chromatograms obtained by the analysis of the seven calibration standards.

RESULTS

In this work, an Ascentis^® Express C18 HPLC column was utilized for the HPLC-ELSD analysis of polysorbate 80 in two different batches of an Erbitux^® antibody drug formulation. The HPLC column applied is comprised of reversed phase-modified, superficially porous silica particles and enables a fast, high-performance analysis. Sample preparation by hydrophilc-lipophilic balanced solid phase extraction was shown to effectively separate PS 80 from major amounts of drug product excipients. Duplicates of a total of five samples of each of the batches B4G and BM9 were analyzed to determine their polysorbate 80 content (see also Table 5). The corresponding ELSD traces of two representative samples of each batch are shown in Figure 4.
The analysis results revealed a PS 80 content of the samples of 0.12 and 0.14 mg/mL, which is in line with typical surfactant concentrations in antibody drug formulations. The calibration curve displayed an excellent quadratic fit over the entire calibration range, with an R2 value of 0.9997, and the LOD for the HPLC-ELSD method was 0.0055 mg/mL.

CONCLUSION

This report describes the entire workflow for the quantitative analysis of polysorbate 80 in two Erbitux^® antibody drug formulations, using reversed phase HPLC-ELSD analysis. An Ascentis^® Express C18 HPLC column packed with superficially porous silica particles was applied for the separation of PS 80 and matrix compounds.

The workflow includes a sample purification process using solid phase extraction with HLB cartridges and subsequent analysis of the samples by reversed-phase HPLC-ELSD. HPLC system calibration data was obtained by the preparation and analysis of seven polysorbate 80 standard solutions and allows for a simple quantification of polysorbate 80 content in mAb samples.

The chromatographic method established is suitable for sample separation and analysis of PS 80 and can also be applied in the quantification of similar non-ionic surfactants.