Quantification of an intact monoclonal antibody, rituximab, by (RP)HPLC/DAD in compliance with ICH guidelines

Abstract

We studied the quantification of an intact therapeutic monoclonal antibody (mAb), rituximab (RTX), using (reversephase) high-performance liquid chromatography with diode array detection ((RP)HPLC/DAD). To this end, we developed a chromatographic method and validated it as stabilityindicating in accordance with the International Conference on Harmonization guidelines (ICH). A 300-Å C8 column (250 mm×4.6 mm, 5 μm) was used to perform the analysis, and the temperature was maintained at 70 °C. Although only one mAb was analyzed, it was necessary to apply a gradient to elute it with a complex organic mixture. Chromatograms were registered at several wavelengths, with λ =214 nm employed for quantification purposes. The method was developed to quantify marketed RTX under typical hospital administration conditions. Further dilution was avoided in order to prevent additional mAb modification, and in this way the method was shown to be linear from 60 to 5000 mg/L. The precision of the method (repeatability and intermediate precision, estimated as the relative standard deviation, RSD %), was less than 1.0 %. Accuracy, specificity, robustness, and system suitability were also evaluated as specified in the ICH guidelines.We conducted a comprehensive chromatographic analysis by submitting RTX to several informative stress conditions. These forced degradation studies were conducted for two reasons: to estimate the specificity of the method, and to evaluate the robustness of the mAb formulation against external stress factors when handling it in preparation for administration. Thus, we investigated the effects of acid, base, oxidation, ionic strength, temperature, and UV light. Although a slight modification to the intact mAb could not be distinguished chromatographically in the stress studies we conducted, the procedure proposed here to evaluate peak purity enabled us to detect it with a satisfactory level of confidence. The proposed method could therefore be considered stability-indicating for quantyfying the intact mAb since it is qualified to detect its degradation/modification. Finally, the method was used to evaluate RTX in a long-term stability study performed under hospital conditions of use.

The Effects of Light-Accelerated Degradation on the Aggregation of Marketed Therapeutic Monoclonal Antibodies Evaluated by Size-Exclusion Chromatography With Diode Array Detection

“…This behavior was also noted in the other 4 mAbs we studied. Using the similarity test available in the LC system, 41 we studied the similarities between the spectra for the monomers and the aggregates. It is interesting to note that the greatest dissimilarity was observed in the original samples (day 0) of the medicine and in the diluted samples at 5 mg/mL.…”

Validation of Simple Uv-Vis Spectrophotometry Method Based on Ich Q2 (R1) Guideline for the Analysis of Sodium Hypochlorite Using Rhodamine B

“…The objective of the validation is to ensure that an analytical method is suitable for its intended purpose (Silva-Buzanello et al, 2015). The guideline from the International Conference on Harmonization ICH-Q2(R1) clearly describes validation characteristics for consideration in the validation of analytical method (Li, Igne, Drennen, & Anderson, 2016;Navas et al, 2013;Pein, Eckert, Preis, & Breitkreutz, 2013). For the analysis of a impurities, the recommended following characteristics are linearity, range, quantification limit (LOD), repeatability, intermediate precision, accuracy, and specificity (Branch, 2005).…”

Evaluation of the pattern and kinetics of degradation of adalimumab using a stability‐indicating orthogonal testing protocol

“…Several methods have been performed focusing on the verification of physicochemical properties of adalimumab; size exclusion chromatography (SE‐HPLC), cation exchange chromatography (CEX‐HPLC), dynamic light scattering (DLS), turbidimetry, second‐derivative ultraviolet spectrophotometry, Fourier transform infrared spectroscopy and peptide mapping (Navas et al, ; Samra & He, ). To the best of our knowledge, none of these methods has been challenged with forced degradation products of adalimumab and the kinetics of degradation has not been investigated.…”