2.1.4. Similarity Metrics of DM

Although the 1D 1H spectra can be used to assess protein HOS qualitatively, a quantifiable similarity metric is of interest to demonstrate comparability after manufacturing changes or similarity between any two drug brands [38]. Previously, 1D 1H spectra between the reference insulin and the follow-on insulin DPs were chemometrically compared using principal component analysis (PCA) and Mahalanobis distance (DM) metrics, which were derived from PCA space [27]. The previous results on insulin DPs suggested a DM value of 3.3 as the similarity threshold [27], where above 3.3 value there were clear differences in the HOS, while below that there were no discernable differences. Here, the rituximab DPs Rituxan® and Reditux® were compared using the same approach. Excipient free regions of the spectra including the down field amide spectrum are informative for rituximab HOS.

The 1D 1H spectra of seven lots of Rituxan® and three lots of Reditux® (Table S1) were collected using both 850 MHz and 600 MHz spectrometers. Representative spectra from both brands were superimposed and visually appeared similar (Figure 2, left). Spectral regions belonging to excipients, residual solvents, water and blank were excluded before PCA. The PCA results showed that the first two principal components accounted for over 70% of the spectral intensity variations (Figure 2, right). The Mahalanobis distance (DM) calculated from the first three principal components (Table S2) using Equations (1) and (2) were 1.95 and 3.15, when the 850 MHz data and the 600 MHz data were used, respectively. Both values were below the established similarity threshold DM value of 3.3 [27], suggesting similar HOS between the two products. Ninety percent (90%) confidence interval ellipses were drawn for the Rituxan® DP lots in PC1/2 space (Figure 2, right). For the 850 MHz data, two lots of Reditux® fell outside the ellipse; for the 600 MHz data, one lot of Reditux® fell outside the ellipse. Therefore, the slight difference in field dependent DM values is not necessarily correlated to the apparent differences shown in the ellipse circles. The results suggest any DM values below the metric of 3.3 would indicate high similarity.
