Date recorded: October 26 2016

Duration: 45 minutes 24 seconds

This webinar will describe the use of Isothermal Titration Calorimetry (ITC) to establish the enzymatic properties of a lysosomal enzyme with potential therapeutic value. The technique was used by the team at Swedish Orphan Biovitrum (Sobi) to enable the evaluation of multiple substrates, and to determine basic enzymatic characteristics. Dr. Stefan Svensson Gelius will describe how, in initial experiments, ITC enabled the demonstration of adhesion of the enzyme to surfaces, and later allowed confirmation of optimal buffer conditions. He will also discuss the problems the team encountered due to the polydispersity of a complex substrate mixture.

The ITC methods developed at Sobi have been used to characterize batches and to establish structure-activity relationships supporting the development of the enzyme as a therapeutic molecule.
Table of contents
1. Welcome
00:13
2. Introductions
01:39
3. Using Isothermal Titration Calorimetry to support the development of therapeutic enzymes
00:15
4. Sobi is an international specialty healthcare company
00:21
5. Untitled
00:28
6. Therapeutic enzymes and disease- Lysosomal storage diseases (LSDs)
02:54
7. Enzymatic activity is a cardinal quality attribute in the release specification of these drug products
01:13
8. Example: Lysosomal enzymes that degrade heparan sulphate in the lysosome
01:11
9. Example: Lysosomal enzymes that degrade heparan sulphate in the lysosome- heparan sulphate oligomers transported to lysosome and degraded stepwize from the non-reducing end
00:29
10. Sequential degradation of heparan sulfate
01:09
11. Aims with study
00:45
12. Potential substrates
01:27
13. Conventional way of developing an end point enzymatic assay
02:02
14. Conventional way of developing an end point enzymatic assay that is coupled to a second developing reaction
00:05
15. Basics of the isothermal titration calorimetry experiment
00:57
16. Basics of the isothermal titration calorimetry experiment
00:10
17. Basics of the isothermal titration calorimetry experiment
00:32
18. Basics of the isothermal titration calorimetry experiment
00:03
19. Basics of the isothermal titration calorimetry experiment
00:16
20. Setting up iTC experiment with tetra-saccharide substrate 1) Quality of substrate?
02:00
21. Setting up iTC experiment2) Determine ΔH
00:05
22. Setting up iTC experiment2) Determine ΔH
00:26
23. Setting up iTC experiment2) Determine ΔH
00:11
24. Setting up iTC experimentBenefits by doing multiple ΔH determinations in same experiment
00:47
25. Setting up iTC experimentBenefits by doing multiple ΔH determinations in same experiment
01:26
26. Setting up iTC experimentBenefits by doing multiple ΔH determinations in same experiment
01:09
27. Setting up iTC experiment3) Determine rate as a function of [S]
01:07
28. Setting up iTC experiment3) Determine rate as a function of [S]
01:30
29. Setting up iTC experiment3) Determine rate as a function of [S]
01:05
30. Results with tetrasaccharide substrate
01:00
31. Results with tetrasaccharide substrate
01:58
32. Calorimetric assay with surrogate substrate- followed same assay setup as with tetrasaccharide substrate
01:49
33. Calorimetric assay with surrogate substrate - followed same assay setup as with tetrasaccharide substrate
00:23
34. Calorimetric assay with surrogate substrate
01:16
35. Good agreement between spectroscopic and calorimetric assaywith surrogate substrate
00:57
36. More challenging substrates- polydisperse large Mw fraction of heparin
02:25
37. More challenging substrates- polydisperse large Mw fraction of heparin
01:25
38. More challenging substrates- polydisperse large Mw fraction of heparin
00:25
39. More challenging substrates- polydisperse large Mw fraction of heparin
00:43
40. Some basic conclusions
00:25
41. Pros and cons with iTC
01:32
42. Pros and cons with iTC
01:24
43. Thank You for Attending
00:21
44. Thank you for your attentionAny questions?
03:03
45. Contact Information
00:23