Using Isothermal Titration Calorimetry to support the development of therapeutic enzymes

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00:00:00 Welcome
00:00:13 Introductions
00:01:52 Using Isothermal Titration Calorimetry to support the development of therapeutic enzymes
00:02:07 Sobi is an international specialty healthcare company
00:02:28 Untitled
00:02:56 Therapeutic enzymes and disease- Lysosomal storage diseases (LSDs)
00:05:50 Enzymatic activity is a cardinal quality attribute in the release specification of these drug products
00:07:03 Example: Lysosomal enzymes that degrade heparan sulphate in the lysosome
00:08:14 Example: Lysosomal enzymes that degrade heparan sulphate in the lysosome- heparan sulphate oligomers transported to lysosome and degraded stepwize from the non-reducing end
00:08:43 Sequential degradation of heparan sulfate
00:09:52 Aims with study
00:10:37 Potential substrates
00:12:04 Conventional way of developing an end point enzymatic assay
00:14:06 Conventional way of developing an end point enzymatic assay that is coupled to a second developing reaction
00:14:11 Basics of the isothermal titration calorimetry experiment
00:15:08 Basics of the isothermal titration calorimetry experiment
00:15:18 Basics of the isothermal titration calorimetry experiment
00:15:50 Basics of the isothermal titration calorimetry experiment
00:15:53 Basics of the isothermal titration calorimetry experiment
00:16:09 Setting up iTC experiment with tetra-saccharide substrate 1) Quality of substrate?
00:18:09 Setting up iTC experiment2) Determine ΔH
00:18:14 Setting up iTC experiment2) Determine ΔH
00:18:40 Setting up iTC experiment2) Determine ΔH
00:18:51 Setting up iTC experimentBenefits by doing multiple ΔH determinations in same experiment
00:19:38 Setting up iTC experimentBenefits by doing multiple ΔH determinations in same experiment
00:21:04 Setting up iTC experimentBenefits by doing multiple ΔH determinations in same experiment
00:22:13 Setting up iTC experiment3) Determine rate as a function of [S]
00:23:20 Setting up iTC experiment3) Determine rate as a function of [S]
00:24:50 Setting up iTC experiment3) Determine rate as a function of [S]
00:25:55 Results with tetrasaccharide substrate
00:26:55 Results with tetrasaccharide substrate
00:28:53 Calorimetric assay with surrogate substrate- followed same assay setup as with tetrasaccharide substrate
00:30:42 Calorimetric assay with surrogate substrate - followed same assay setup as with tetrasaccharide substrate
00:31:05 Calorimetric assay with surrogate substrate
00:32:21 Good agreement between spectroscopic and calorimetric assaywith surrogate substrate
00:33:18 More challenging substrates- polydisperse large Mw fraction of heparin
00:35:43 More challenging substrates- polydisperse large Mw fraction of heparin
00:37:08 More challenging substrates- polydisperse large Mw fraction of heparin
00:37:33 More challenging substrates- polydisperse large Mw fraction of heparin
00:38:16 Some basic conclusions
00:38:41 Pros and cons with iTC
00:40:13 Pros and cons with iTC
00:41:37 Thank You for Attending
00:41:58 Thank you for your attentionAny questions?
00:45:01 Contact Information
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.

Présentateur

Stefan Svensson Gelius is a Principal Scientist at the Division of Research and Translational Science at Sobi (Swedish Orphan Biovitrum) in Sweden, focusing on innovations in the biopharmaceutical field. Stefan has previously worked within the structural biology teams at Pharmacia and Biovitrum and has a background in enzymology from the Karolinska Institutet in Stockholm.