| 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.
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.
발표자
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.