10 November 2012

Article Review – Long-term stability of diluted solutions of the monoclonal antibody rituximab

Full Article Title – Muriel Paul, Victoire Vieillard, Emmanuel Jaccoulet, Alain Astier. Long-term stability of diluted solutions of the monoclonal antibody rituximab. International Journal of Pharmaceutics (2012) 436, pp 282– 290

Overview

This article represents a good piece of academic research to investigate changes to the chemical, physical and functional attributes of Rituximab upon long-term storage as a diluted solution. The approach taken in the article to identify changes upon storage is well considered, as the authors appreciate and explicitly state the fundamental requirements for the analytical protocols to evaluate primary, secondary, tertiary and quaternary structure for a comprehensive characterization of chemical/physical stability, as well the need to utilize cell-based methods for evaluating functional activity.

Perhaps most importantly though, the article represents an excellent example of when care and diligence must be exercised in ‘interpreting’ an academic study, particularly when one seeks to consider applying its conclusions to extending the shelf-life of a real product. Although this article stands as a good piece of academic research, it does not attempt to address several key regulatory requirements, nor would these ever be considered during the peer-review process for an academic article.

Here, we briefly describe just a couple of examples where results from this study would not be considered sufficient for their use in applying an extended shelf-life to Rituximab for clinical use.

Methodology

The article conducts all stability testing onRituximab samples stored at only a single concentration (1 mg/mL). It is recommended (ICH Q5c) that stability testing must be carried out at concentrations representative of the final product, which in the case of Rituximab, would be over a wide range of concentrations as stated in the SPC (1-4 mg/mL).

Turbidity

Turbidity was the only technique used in the article to characterize larger aggregates and insoluble precipitates, so stands as the only method of characterizing and quantifying particulate levels. Strict limits for particle numbers with size ranges ≥10 micron and ≥25 micron are applied in the BP and EP to injectibles, including monoclonal antibodies.

  • Unfortunately, complete primary data was not presented in the article detailing the turbidity results, rather a comment in the text that no change was observed.
  • It is also stated in the methods section (outlining details for each protocol) that samples for UV analysis were CENTRIFUGED PRIOR TO ANALYSIS. This process would be likely to influence the results from turbidity measurements, likely reducing the ability to observe larger particles.
  • The above point is highlighted by the fact that the samples stored at 40˚C for 6 months show significant levels of degradation according to some other techniques, but are still quoted as showing almost no changes in the turbidity assay as performed. It has been shown previously that changes to levels of aggregation detected through alternative techniques should be reflected in significant changes to turbidity levels. [1]
  • Finally, the turbidity technique is not a quantitative method, so NO actual particle numbers or sizes are obtained and, hence, BP particle limits can not be applied.

Functional Activity

For the purpose of assigning an extended shelf life, a suitable assay to determine functional activity would need to demonstrate it was unlikely that any significant change in clinical efficacy has occurred as a result of prolonged storage. This article describes the use of a cell based assay for measuring direct cytotoxicity of Rituximab towards the Raji cell line, as its measure of functional activity.

  • It is known that direct cytotoxicity (as evaluated in the article) is NOT a pathway through which Rituxmab exerts its clinical effects in vivo, and so the results detailed in the article tell little about changes that would be expected in clinical efficacy. [2]
  • The paper presents the activity results in the form of IC50, IC25 and AUC (area under the curve) values. All three values are generated from the same data, and so actually all represent the same thing, not 3 independent measures of activity.
  • Determining activity in this format (IC50, IC25 and AUC) is unlikely to provide the sensitivity necessary for identifying minor changes in activity (±5-10%) over a prolonged period of time. In this article, Raji cells are incubated with Rituximab over a large range of concentrations, from very low concentrations (where the S.D.’s for individual assays are ±70%, hence changes of ±5% in activity will not be seen) to very high concentrations (where there is so much Rituximab present that a small loss of activity would not be detected as a result of receptor saturation).

References

[1] Hanns-Christian Mahlera, Robert Mullera, Wolfgang Friebb, Aurelie Delillea, Susanne Matheusa, European Journal of Pharmaceutics and Biopharmaceutics (2005) 59, 407–417

[2] Glennie MJ, French R, Cragg MS, and Taylor RP: Mechanisms of killing by anti-CD20 monoclonal antibodies. Mol. Immunol. (2007) 44, 3823-3837.