Date recorded: March 14 2019

Duration: 43 minutes 14 seconds

Therapeutic proteins are miracle drugs for millions of patients globally. Unfortunately, with many of these products, a relatively large fraction of patients experiences loss of efficacy due to adverse immunogenicity. Studies for more than 50 years in humans and in animal models have documented that aggregates and particles are important causes of immunogenicity. Also, aggregate and particle levels are critical quality attributes for protein products. Every step in the manufacturing, shipping and use of a protein product can cause aggregation and particle formation, from initial fermentation to final delivery to patients. Developing effective control strategies requires insights into mechanisms for aggregate and particle formation, as well as sensitive and robust methods for characterizing and quantifying soluble aggregates, nanoparticles and microparticles. In bulk solution, aggregates are formed from partially unfolded protein molecules, and aggregation rate is modulated by protein conformational and colloidal stabilities. Often, choosing an optimum pH will help to minimize aggregation by favoring native state stability and increasing charge-charge repulsion between protein molecules.

However, even with an optimized formulation, which minimizes aggregation in bulk solution, interfacial stresses can readily cause protein particle formation. Protein molecules adsorb to interfaces (e.g., air-water, ice-water and water-solid), aggregate and form films. Mechanical rupture of these films releases protein particles into the bulk solution. Such film formation and rupture is common throughout a product’s life history. Fully understanding the causes and control of surface-mediated particle formation requires robust characterization of nano- and microparticles. As an example, this presentation will show results for commercial filling pump operation using a peristaltic pump and three different brands of commercially-used tubing. One highlight from the study was that pumping a protein solution through Pharmed tubing resulted in much lower microparticles levels than pumping through Accusil or Masterflex tubing. But nanoparticle levels in samples pumped through Pharmed tubing were much higher than those observed in samples pumped through the other tubing brands. Furthermore, with an accelerated degradation method of post-pumping agitation, it was seen that the high level of nanoparticles resulted in very high concentrations of microparticles. Effects of formulation pH (affecting conformational and colloidal stabilities) and surfactants were also tested, and results indicated that both nano- and microparticle measurements are crucial for understanding fully the impact of these solution conditions. Finally, light obscuration was also used to measure microparticles, and it was found that this method was not suitable to detect and quantify increases in protein particles resulting from filling pump operation or post-pumping agitation.

Table of contents
1. Aggregates and particles in therapeutic protein products: Causes, characterization and control
02:11
2. Aggregates and particles in therapeutic protein products: Causes, characterization and control
00:36
3. Acknowledgments
00:06
4. Adverse Immunogenicity: When Miracle Drugs Fail
00:23
5. Immunogenicity: Particles as Adjuvants
00:35
6. Required reading
00:20
7. Untitled
01:32
8. Causes of Protein Aggregation
01:17
9. Untitled
01:21
10. Why do subvisible protein particles so often look like the ones of the left?
00:41
11. Causes of Protein Particles: Interfaces
01:15
12. Particles going along for the ride
01:14
13. Particle Generation during Pumping and Post-pumping Agitation
00:28
14. Objectives
00:31
15. Untitled
01:05
16. Comparison of Nanoparticle Concentration during 500 mL Fill Volume of 1 mg/mL IVIG in PBS via Nanoparticle Tracking Analysis (Nanosight NS300)
00:45
17. Comparison of Nanoparticle Concentration during 500 mL Fill Volume of 1 mg/mL IVIG in PBS via Resonant Mass Measurement (Archimedes)
00:45
18. Effects of Post-Pumping Agitation
02:25
19. Particulate Formation of IVIG Pumped in Different Buffers
00:26
20. Why PBS and Glycine?
00:43
21. Comparison of Microparticle Concentration during 500 mL Fill Volume of 1 mg/mL IVIG in Different Buffers via Flow Microscopy (FlowCamTM)
00:32
22. Comparison of Nanoparticle Concentration during 500 mL Fill Volume of 1 mg/mL IVIG in Different Buffers via Nanoparticle Tracking Analysis (Nanosight NS300)
00:08
23. Comparison of Nanoparticle Concentration during 500 mL Fill Volume of 1 mg/mL IVIG in Different Buffers via Resonant Mass Measurement (Archimedes)
00:40
24. PBS versus Glycine Buffer: Post-Pumping Agitation
00:05
25. Comparison of Microparticle Concentration during 500 mL Fill Volume of 1 mg/mL IVIG in Different Buffers via Flow Microscopy (FlowCamTM)
00:38
26. Comparison of Nanoparticle Concentration during 500 mL Fill Volume of 1 mg/mL IVIG in Different Buffers via Nanoparticle Tracking Analysis (Nanosight NS300)
00:18
27. Comparison of Nanoparticle Concentration during 500 mL Fill Volume of 1 mg/mL IVIG in Different Buffers via Resonant Mass Measurement (Archimedes)
00:40
28. Effect of Polysorbate 80
00:21
29. Comparison of Microparticle Concentration for 500 mL Fill Operation of 1 mg/mL IVIG in PBS with PS80 using Various Tubing via Flow Microscopy
00:16
30. Comparison of Nanoparticle Concentration for 500 mL Fill Operation of 1 mg/mL IVIG in PBS with PS80 using Various Tubing via Nanoparticle Tracking Analaysis
00:11
31. Comparison of Nanoparticle Concentration for 500 mL Fill Operation of 1 mg/mL IVIG in PBS with different PS80 concentrations using Various Tubing via Resonant Mass Measurement (Archimedes)
00:30
32. Post-Pumping Agitation in the Presence of Surfactant
00:05
33. Time Dependent Comparison of Micro and Nanoparticle Concentration for 500 mL Fill Operation for Agitated 1 mg/mL IVIG in PBS in the presence of 0.02% PS80 with Various Tubing
01:14
34. Comparison of FlowCam to Light Obscuration
00:12
35. FlowCamTM versus HIAC 9703+ of 1 mg/mL IVIG in PBS During Pumping Run
00:54
36. FlowCamTM versus HIAC 9703+ in PBS Post Pumping Agitation
01:55
37. Both nano- and microparticles must be monitored to develop effective control strategies
02:29
38. 2019 Colorado Protein Stability Conference: 25th Anniversary
00:25
39. Thank you for your attentionQuestion & Answer SessionListening live:Ask your question by typing within the Q & A chat facility Listening on-demand:Send your questions toevents@malvernpanalytical.com
13:02