Date recorded: September 25 2014

Duration: 52 minutes 49 seconds

Recent work by our guest presenter, Dr. John Carpenter (University of Colorado Anschutz Medical Center), discovered that nanoparticles present in solutions of intravenous immunoglobulin (IVIG) serve as precursors for microparticles during pharmaceutically-relevant stresses such as freeze-thawing or agitation. Depletion of nanoparticles (e.g. by ultracentrifugation) prior to stress greatly reduces the rate of microparticle formation. However new nanoparticles can form during the stress.

Also, therapeutic proteins can adsorb to foreign nanoparticles, resulting in particles containing both protein and foreign material. Sources of nanoparticles of foreign materials include filters, filling and transfer pumps, and chromatography columns.

Importantly, nanoparticles of therapeutic proteins (with either protein alone or with foreign materials) can induce adverse immunogenicity. Our recent study of mouse growth hormone in mice documented that samples containing only protein monomers and trace amounts of nanoparticles were highly immunogenic when administered subcutaneously or intravenously.

Taken together, current data show that nanoparticles play important roles in protein aggregation pathways and in adverse immunogenicity. Thus, regulatory agencies are now viewing quantitation and sizing of nanoparticles as important parts of product quality assessment.

Table of contents
1. Roles of Nanoparticles in Aggregation Pathways, Adverse Immunogenicity and Quality Assessment of Therapeutic Proteins
01:32
2. NTA history
01:47
3. NanoSight Technology: How does it work?
00:35
4. Nanoparticle Tracking Analysis (NTA) – Direct Visualization of Nanoparticles
00:00
5. Nanoparticle Tracking Analysis (NTA)
00:00
6. Principle of Measurement
00:26
7. Sizing: Stokes-Einstein
00:00
8. Concentration: Particles are Counted by Number
00:36
9. NTA Detection Limits: Size and Concentration
01:06
10. NTA Parameters measured – simultaneously, ‘real time’, particle-by-particle...additional benefit
01:18
11. NanoSight Technology
00:49
12. Roles of Nanoparticles in Aggregation Pathways, Adverse Immunogenicity and Quality Assessment of Therapeutic Proteins
01:48
13. Acknowledgments
00:18
14. Outline
00:25
15. You never know what you might find until you look, with proper tools
00:30
16. Causes of Protein Aggregation
00:36
17. New Insights into Causes of Aggregation from Quantifying Subvisible Particles
00:17
18. Particle counts (MFI) provide highly sensitive measure of aggregation due to freeze-thawing
01:11
19. Particles going along for the ride
00:48
20. Stainless steel nanoparticles shed from filling pump “seed” protein microparticles
00:12
21. Particle Formation during Vial Filling with an IgG: Early Example
00:28
22. Positive Displacement Piston Pumps
00:25
23. Particle-containing Solution from Commercial Vials was Filtered and Repumped
00:23
24. Particle Shedding during Pumping of Buffer without Protein
00:08
25. Material Wiped from Piston after 15-ml Pumping Cycle with Buffer
00:11
26. Nanoparticles Shed from Pump
00:15
27. Foreign Material in Pumped Buffer Induces
00:33
28. Freeze-Thawing IVIG with & without Prior Centrifugation: Nanoparticles
01:07
29. Freeze-Thawing IVIG with and without prior ultracentrifugation: MFI Results
00:37
30. Role of Nano- and Microparticles on IVIG Particle Formation during Agitation
00:17
31. Microparticle Concentrations after Initial Agitation and Subsequent Centrifugations
00:13
32. Nanoparticle Concentrations after Initial Agitation and Subsequent Centrifugation
00:15
33. Microparticles during agitation
00:45
34. Nanoparticle Concentrations during Agitation
00:40
35. Subvisible Particles are Critical Species on the Protein Aggregation Pathway
00:37
36. Adverse Immunogenicity: When Miracle Drugs Fail
01:26
37. Immunogenicity: Particles as Adjuvants
00:23
38. Effects of route of administration on immunogenicity of rmGH
00:16
39. Particles in rmGH Samples: only nanoparticles in ultracentrifuged sample
00:47
40. Ultracentrifuged rmGH (week 6): Nanoparticles cause Immunogenicity
00:59
41. Mishandling of Products by Pharmacies, Clinics and Patients
00:37
42. Stresses to Protein Therapeutics even during “Proper” Handling for IV Infusion
01:42
43. Mimicking the infusion setup…
00:21
44. Saline - particle counts > 1µm
00:30
45. Saline – total nanoparticle counts
00:35
46. IVIg in Saline – particle counts > 1µm
00:16
47. IVIg in Saline – total nanoparticle counts
00:37
48. IVIg-0.22um- filter FlowCam Images
00:33
49. Polycarbonate Particles in IV Saline: G3 ID Raman Microspectroscopy
01:17
50. Current Regulatory Expectations for Analysis of Subvisible Particles
02:20
51. Conclusions
01:17
52. 2015 Colorado Protein Stability Conference
17:00
53. Contact Information
00:45