Methods for assessing cell surface glycosylation

1. A method for assessing cell surface glycans, the method comprising:

(a)(i) incubating a test composition comprising a plurality of cells under conditions to release one or more glycans from the surface of cells in the test composition, wherein a sample comprising one or more cell surface glycans is generated and (a)(ii) determining the presence, absence, identity and/or level of glycans present in the sample, thereby assessing the cell surface glycan profile of the sample; or

(b) determining the presence, absence, identity and/or level of glycans present in a sample, thereby assessing the cell surface glycan profile of the sample, wherein the sample comprises one or more glycans released from the surface of cells present in a test composition comprising a plurality of cells after incubation of the test composition under conditions to release the one or more glycans; and

wherein the cells express a recombinant receptor or the test composition comprises cells expressing a recombinant receptor.

2. The method of claim 1 , wherein cells in the test cell composition comprise whole cells or intact cells.

3. The method of claim 1 , wherein the test cell composition comprises between 1×10 6 cells and 5×10 6 cells, inclusive.

4. The method of claim 1 , wherein the test cell composition comprises a concentration of between 1×10 5 cells/mL and 1×10 8 cells/mL, inclusive.

5. The method of claim 1 , wherein the incubation is carried out in the presence of an N-glycosidase.

6. The method of claim 5 , wherein the N-glycosidase is a peptide N-glycosidase (PNGase) F.

7. The method of claim 6 , wherein the PNGase F is recombinant.

8. The method of claim 6 , wherein the PNGase F comprises a PGNase F of Flavobacterium meningosepticum , or a portion or mutant thereof that is enzymatically active.

9. The method of claim 6 , wherein the PNGase F comprises an amino acid sequence that exhibits at least 85% or more sequence identity to SEQ ID NO:1 or is a portion thereof that is enzymatically active.

10. The method of claim 6 , wherein the PNGase F comprises the amino acid sequence set forth in SEQ ID NO: 1.

11. The method of claim 6 , wherein the amount of PNGase F is 1 unit to 5000 units, inclusive.

12. The method of claim 1 , wherein the incubating the test composition is for an amount of time that is between or between about 5 minutes and 12 hours, inclusive.

13. The method of claim 1 , wherein the incubating the test composition is at a temperature between 25° C. and 39° C.

14. The method of claim 1 , wherein, prior to the determining the presence, absence, identity and/or level of glycans present in a sample, the method further comprises labeling glycans from the sample with a detectable label.

15. The method of claim 14 , wherein the detectable label is a fluorescent label and the fluorescent label is or comprises 2-aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA), 2-aminopyridine (PA), 2-Aminoacridone (AMAC), 2-aminonaphthalene trisulfonic acid (ANTS), 1-aminopyrene-3,6,8-trisulfonic acid (APTS), 3-(Acetylamino)-6-aminoacridin (AA-Ac), 6-Aminoquinoline (6-AQ), 7-Aminomethyl-coumarin (AMC), 2-Amino (6-amido-biotinyl) pyridine (BAP), 9-Fluorenylmethoxycarbonyl (FMOC)-hydrazide, 1,2-Diamino-4,5-methylenedioxy-benzene (DMB), or o-Phenylenediamine (OPD).

16. The method of claim 14 , wherein the detectable label is a fluorescent label comprising a carbamate tagging group, a quinolone fluorophore, and a tertiary amine.

17. The method of claim 1 , wherein, prior to determining the presence, absence, identity and/or level of the one or more glycans, the sample is subjected to glycan purification or enrichment.

18. The method of claim 1 , wherein determining the presence, absence, identity and/or level of the one or more glycans comprises subjecting the sample to mass spectrometry.

19. The method of claim 1 , wherein determining the presence, absence, identity and/or level of glycans comprises subjecting the sample to liquid chromatography (LC) followed by mass spectrometry.

20. The method of claim 19 , wherein the liquid chromatography is hydrophilic interaction chromatography (HILIC).

21. The method of claim 18 , wherein the mass spectrometry comprises ESI-MS or-tandem mass spectrometry (MS/MS).

22. The method of claim 1 , wherein the determining the presence, absence, identity and/or level of the one or more glycans comprises analyzing one or more glycan structure or structures for branching, linkages between monosaccharides and/or location of monosaccharides.

23. The method of claim 1 , wherein the one or more glycans comprises high mannose N-glycans, bisected and Sialyl Lewis X N-glycans, and/or N-acetyl lactosamine containing N-glycans.

24. The method of claim 1 , wherein the cells comprise stem cells, immune cells, white blood cells, peripheral blood mononuclear cells (PBMC), lymphocytes, or unfractionated T cells.

25. The method of claim 1 , wherein the cells comprise T cells that are CD4+ and/or CD8+ T cells or the test cell composition comprises T cells that are CD4+ and/or CD8+ T cells.

26. The method of claim 1 , wherein the recombinant receptor is or comprises a chimeric receptor and/or a recombinant antigen receptor.

27. The method of claim 1 , wherein the recombinant receptor is or comprises a T cell receptor or a chimeric antigen receptor (CAR).

28. The method of claim 1 , wherein:

the test cell composition comprises a concentration of between 1×10 6 cells/mL and 5×10 7 cells/mL, inclusive; or

the incubation is carried out in the presence of a PNGase F comprising the amino acid sequence set forth in SEQ ID NO: 1 or a portion or mutant thereof that is enzymatically active; or

the incubating the test composition is for an amount of time that between or between about 30 minutes and 6 hours, inclusive; or

the incubating the test composition is at a temperature between 35° C. and 39° C.