Enzyme replacement therapy represents promising treatments especially for lysosomal storage diseases (deficiency in an enzyme required for the lysosomal function). As Mannose 6-phosphate (Man 6-P) serves as a targeting signal for lysosomal enzymes, the amount of Man 6-P (expressed as mol Man 6-P/mol protein) is an important identity test.  It is directly related to the potency of the product because it insures an effective intracellular localization. Quantification of Man 6-P is required as a release assay and as a stability indicating assay during long term stability studies.

The current quantification of Man 6-P content in glycoproteins requires the hydrolysis of the glycans under acidic conditions (acetic acid or trifluoroacetic acid 2-4 M at 80-100 °C for several hours, generally 2-4 hours). The monosaccharides obtained after hydrolysis are separated using anion exchange chromatography and detected by pulsed amperometry or by MS. [1] Common problems associated with acid hydrolysis are incomplete glycan cleavage and instability of the released monosaccharides under acidic conditions. [2]

We have now demonstrated the possibility to use FTIR spectroscopy to evaluate the content of Man 6-P in glycoprotein. FTIR is an ideal tool as protein bands and glycan bands appear in distinct areas of the spectrum. Based on a Partial Least Square procedure, a predictive model able to evaluate the Man 6-P content was calibrated. In the figure hereunder, the predicted values are represented as a function of the true values. The correlation coefficient between these values is very close to 1, indicating a high performance of the model.

Figure M6P + legendThe quantification of Man 6-P through FTIR spectroscopy has two main advantages:

  • It is performed on the whole glycoprotein. Sample preparation is thus very limited as no hydrolysis/separation/MS is required. This also avoids errors due to the variability in the efficiency of the hydrolysis.
  • Once the predictive model is validated, the processing time is much shorter than with the current method described above. A FTIR spectrum is obtained in 2 minutes, the analysis takes a few seconds.

 

References:

[1]        A. Planinc, J. Bones, B. Dejaegher, P. Van Antwerpen, C. Delporte, Glycan characterization of biopharmaceuticals: Updates and perspectives, Anal. Chim. Acta. 921 (2016) 13–27. doi:10.1016/j.aca.2016.03.049.

[2]        E. Higgins, Carbohydrate analysis throughout the development of a protein therapeutic., Glycoconj. J. 27 (2010) 211–25. doi:10.1007/s10719-009-9261-x.