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.  Common problems associated with acid hydrolysis are incomplete glycan cleavage and instability of the released monosaccharides under acidic conditions. 
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.
The 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.
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 E. Higgins, Carbohydrate analysis throughout the development of a protein therapeutic., Glycoconj. J. 27 (2010) 211–25. doi:10.1007/s10719-009-9261-x.