Recommended
Recommended
More Related Content
What's hot
What's hot (19)
Viewers also liked
Similar to Protein Aggregation Properties Revealed by New Methods
Similar to Protein Aggregation Properties Revealed by New Methods (20)
Protein Aggregation Properties Revealed by New Methods
- 1. Aggregation Properties of Therapeutic Proteins Revealed by New Analytical Methods Danny K. Chou, PharmD, PhD President, Compassion BioSolution, LLC Biologics world Taiwan 2016 25th, February 2016
- 2. Presentation Outline • How protein aggregation occurs and the dominant forces that control the formation of aggregates and particulates. • How does protein aggregation affect development and commercial viability of biopharmaceuticals • How do you monitor and control formation of aggregates and particles? What is the state-of the- art analytical approach? • Why proper integration of formulation, container- closure, and analytical technology is essential to the success of a biologics development program.
- 3. Success Drivers in Biologic Drug Development • Thanks to the favorable clinical profile of biologics and increasing market demand the growth in development of biopharmaceuticals is already surpassing that of conventional drugs. • Along with this trend the challenges of biopharmaceutical development has become a significant barrier to entry and sustainable commercial success • Commercial success requires both innovative technical development and management of unique challenges associated with the nature of biologics • One of the these key technical challenges is protein aggregation
- 4. Why Are Proteins so Difficult to Develop? Putting Things Into Perspective With Respect to Size of Biologic Molecules *NEJM 2011
- 5. What is Protein Aggregation and Why is it Important? • Protein aggregates: “High molecular weight proteins composed of multimers of natively conformed or denatured monomers” (Rosenberg, 2006) • Aggregates can reduce biological activity, or worse, induce immune response, but the mechanism is still not very well understood • Immunogenicity is a major product SAFETY concern
- 6. Protein Aggregation – Mechanisms • Protein therapeutics are highly complex in terms of size, structure and function. • Structural flexibility presents a higher risk for physical instability as well as a major regulatory concern on product quality and safety. Krishnamurthy et al. BioProcess International, 2008
- 7. Phenomenon of Protein Aggregation – What Do We Know at the Present? • Both conformational and colloidal stability play a role Chi et al., Pharm. Res. 20:1325, 2003
- 8. Contributing Factors to Formation of Soluble Protein Aggregates and Particles Bioprocessing from start to end Physical/chemical Stresses: pH, ionic strength, temperature, chemical modification, light, agitation, mechanical shock, freeze- thaw, etc. Air/Solid-Liquid Interfaces: Protein contact with tubings, pumps, pipes, vessels, filters, columns, etc. Foreign Particles: Stainless steel, glass, plastic, rubber, tungsten, silicone oil, etc. Fermentation/cell culture Purification Filling Packaging Shipping Storage Administration
- 9. Not all Aggregates or Particles are the same…. P G P G P G P G Dissociable Dimer P G P G P G P G Non Dissociable Dimer P G P G P G P G P G P G P G P G P G P G Non Dissociable Aggregate P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G Subvisible Particles P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G P G Visible Protein Particles Visible Extraneous Particles
- 10. Why is protein aggregation relevant to biopharmaceutical development and manufacturing? • Protein aggregation has long been suspected as a having a role in safety and efficacy of biologics
- 11. How subvisible particles became a key focus for regulators throughout the globe
- 12. Subvisible Particles- a highly visible topic
- 13. Orthogonal Techniques that Cover Various Particle Size Ranges 0.001 um 0.01 um 0.1 um 1 um 10 um 100 um 600 um SEC, AUC DLS Flow microscopy HIAC / Light Obscuration Visual 1 nm 10 nm 100 nm 1000 nm Subvisible aggregates Silicone droplets Nano-emulsions & suspensions Visible aggregates Emulsions & suspensions Glass, rubber, plastic, etc., particles RMM Nanosight
- 14. Extended characterization per USP<1787> Technique Size Range Light obscuration 2-300 um Electrical senzing zone (Coulter) 0.4-1600 um Laser diffraction 0.1-3500 mm Light microscopy 0.3 um to 1 um Flow imaging analysis 1 um-100 um for size distribution; 5-100 um for morphology Electron microscopy (EM): Scanning EM, scanning transmission EM, and transmission EM A to mm Fourier Transform Infrared (FTIR) microspectroscopy 10 um to 1 mm Dispersive-Raman microspectroscopy 0.5 um to 1 mm Electron microscopy (EM) with energy- dispersive X-ray spectrometry (EDS) A to mm for imaging Size and distribution Size and morphology Characterization
- 15. Criteria for Ideal Methodology • Detects SVPs ranging from 0.1 – 100 µmSize Range • Ideal if it allows for validation and setting acceptable limits. Particle Count • Protein Aggregate vs Silicone Oil Droplet vs External Inclusions (Metal, rubber etc.) Particle Type • Recording of particle image; Provision for visual identification and analysis.Image of Particle • Stable Aggregates vs Dilution-dependent Transient aggregates. No prior sample manipulation
- 16. While This Field Continues to Evolve There is an Opportunity in Front of Us • SVP testing can be applied during process development to optimize processing conditions and reduce impurities • Proper integration of orthogonal technique is a powerful way to improve formulation robustness and assess drug product- delivery device compatibility
- 18. Case 1: Use of Subvisible Particulate (SVP) Analysis During Process Development Background: • IgG monoclonal antibody (mAb A) • Manufactured at 2k L scale for Phase I and Phase II clinical testing • 3 Column Purification Platform • Affinity, Cation, Anion • Virus filtration in final position prior to UF/DF
- 19. Validation Study of VF Performance (mAb A) 0 100 200 300 400 500 600 0 200 400 600 VProFlux(LMH) Volumetric Throughput (L/m2) 1% XMuLV Run 1 1% XMuLV Run 2 Decoupled No Virus 1% MVM Run 1 1% MVM Run 2 Coupled No Virus • Achieved only 53% of target throughput • Only non-spiked coupled train (with pre-filter) exceeded target • Decoupled trains displayed both cake formation and pore plugging type fouling • Visible particle formation post transfer of feed into reservoirs • Conclusion: FAIL. Requires revalidation Concentration mg/mL 7.9 *Target Throughput L/m2 (g/m2) > 318 (2510) **Achieved Throughput L/m2 ( g/m2 ) 168 (1330) * Target based on production scale ** Achieved based on worst VF performance
- 20. Key Goals for Viral Filtration Validation Study of mAb A • Overcoming filter fouling – Minimize factors linked to particulate formation • Optimize strategies for handling VF load when conducting VF validation • Measuring sub visible particles (SVP) – Establish SVP analytical techniques and apply to VF loads – Correlating SVP formation and distribution as function of VF handling practices
- 21. Detection Method: Flow Microscopy • Utilizes optical system similar to microscope • Detects particle sizes of 1 μm to >100 mm diameter • Captures real time images of particles in fluid as it passes through a flow cell • Distribute sizes based on equivalent spherical diameter (ESD) or area based diameter (ABD)* *Fluid Imaging Technologies (© 2010). Imaging Particle Analysis – Technology. Retrieved September 4, 2013, from http://www.fluidimaging.com/ EXPERIMENT “Gently” handled VF Load was tested directly on FlowCAM “Non Gentle” handled VF load experienced turbulence (3x pour and swirl) to mimic validation handling practices, then tested on FlowCAM
- 22. FlowCAM Data Acquisition : How is it Done?
- 23. Quantitative Particle Detection of mAb A During Viral Filtration • Particle concentration increased as a result of turbulent, “non gentle” conditions • Handling conditions were linked to membrane fouling; therefore new methods need to be developed to minimize particle formation in an effort to increase process efficiency while reducing cost 222761 61594 7169 4512 2671 9122 1005 69267 7213 2776 1823 946 3582 372 -3.E+04 2.E+04 7.E+04 1.E+05 2.E+05 2.E+05 1-2um 2-4um 4-6um 6-8um 8-10um > 10um > 25um ParticlespermL(P/ML) Particle Size ( μm) Non Gentle Gentle Handling 1-2 μm 2-4 μm 4-6 μm 5-8 μm 8-10 μm >10 μm >25 μm
- 24. Orthogonal Particle Detection Method: DLS Dynamic Light Scattering • DLS (dynamic light scattering, also known as quasi-elastic light scattering), uses light scattering of a laser beam and very fast decay measurement to determine the hydrodynamic radius and polydispersity of the species present. • It can measure radii down to 0.5 nm and determine radii for two populations with radii at least 5-fold different, with best results if 10-fold different Kuebler S. “Characterizing stable protein formulations.” Genetic Engineering & Biotechnology News 27.20 (2007). Accessed September 4, 2013 from http://genengnews.com. EXPERIMENT “Gently” handled VF feed was loaded directly onto microplate for DLS testing “Non Gentle” handled VF load experienced turbulence (3x pour and swirl) to mimic validation handling practices prior to testing Over the duration of one hour, DLS data was collected
- 25. “Gently” handled samples 5-6 nm “Non-Gently” handled samples 5-6 nm Aggregates of mAb • DLS can be used as an orthogonal approach for SVP detection • Detected differences in handling conditions (5-6 nm mAbs vs. large particles) • Non-monomer particles were detected as a result of “non gentle” conditions • 20nm VPro pore size, may be increasingly susceptible to membrane fouling due to handling conditions Qualitative Particle Detection of VF Load
- 26. Impact of Process Change on Process Efficiency and Cost for mAb A • By modifying formulation of in-process material and handling techniques, particle formation was mitigated, enabling flux to maintain > 100 LMH and target throughputs were exceeded • With 1% spikes of both feeds, was able to show adequate log clearance 0 50 100 150 200 250 300 350 400 450 0 100 200 300 400 500 600 VProFlux(LMH) Volumetric Throughput (L/m2) 1% MVM N=1 1% MVM N=2 1% XMuLV N=1 1% XMuLV N=2 Concentration mg/mL 7.0 *Target Throughput L/m2 (g/m2) > 318 (2510) **Achieved Throughput L/m2 ( g/m2 ) 517 (3620) * Target based on production scale ** Achieved based on worst VF performance
- 27. Case 2: Use of SVP Analysis During Combination Product Development • Drug product-container compatibility is a critical factor in the successful development of biologic combination products • SVP testing was conducted to evaluate stability of a high concentration mAb formulation (mAb B), in different brands/types of pre-filled syringes (PFS)
- 28. Glass PFS Plastic PFS Brand Brand X MySafill® Packaging Material Glass (borosilicate) Plastic (Cyclo Olefin Polymer ) Pros Scratch resistant, Transparent Low protein adsorption*, Tungsten not required, Retractable needle, Cons Breakage, Tungsten, Alkali oxide, Negatively charged Surface Higher leachable profile, More easily scratched *In selected cases Glass PFS and MySafill® (a new polymer PFS with integrated safety feature) were directly compared with respect to their impact on stability of mAb B
- 29. What is Different about MySafill®? Same injection technique as the conventional pre-filled syringe; retraction of needle is activated by pressing the plunger rod after completion of injection Courtesy of Medical Chain International
- 30. Shaking Experiment – Visual Observation Active Control Glass PFS A Glass PFS B Glass PFS C MySafill Active Shaken Glass PFS A Glass PFS B Glass PFS C MySafill
- 31. Shaking Experiment – SEC-HPLC 96.0 96.4 96.8 97.2 97.6 98.0 98.4 98.8 %MAINPEAK Control Shaken Glass A Glass CGlass B MySafill
- 32. Shaking Experiment - Flow Microscopy -10000 0 10000 20000 30000 40000 50000 60000 70000 PARTICLECONC.(PARTICLES/ML) Flow Microscopy (Average of 2 Consecutive Runs) Control Shaken Glass PFS A Glass PFS B Glass PFS C MySafill PFS Placebo
- 33. Morphology of Particles is Important for Identification Glass PFS Agitated in bad formulation Glass PFS Agitated in good formulation MySafill Agitated in bad formulation MySafill Agitated in good formulation
- 34. Effect of Stress Method on Aggregate Morphology Images of mechanical stress-induced particles in IgG solution Images of thermal stress-induced particles in IgG solution
- 35. Container Material & Formulation Impact on Subvisible Particle Formation upon Agitation (Flow Microscopy) 0 2000 4000 6000 8000 10000 12000 14000 16000 BD Glass Syringe (no agitation) BD Glass Syringe agitated (No PS 20) Glass Syringe agitated (with PS 20) MySafill (no agitation) MySafill agitated (no PS 20) MySfaill agitated (with PS 20) 2-4um 4-6um 6-8um 8-10um 10-25um greater than 25um ParticleConcentration (particles/mL) Glass, No Agitation Glass-PS, Agitation Glass+PS, Agitation Plastic, No Agitation Plastic-PS, Agitation Plastic+PS, Agitation Proper integration of formulation, delivery device, and analytical technology is essential
- 36. The ‘Triad’ of Biologics Drug Product Development Formulation To Achieve Stability and Process Efficiency New Analytics Enable True Product Characterization Innovative Delivery Technology IV to SC Improved patient care and chance of commercial success Current Situation The Future of Biologics Marketplace The ‘pinnacle’ is not as hard to reach as it may seem
- 37. The Best Selling Brand of a Biologic in Asia Lack of proper integration in biologic drug product development will manifests itself
- 38. Conclusions • New technologies are enabling better understanding of protein aggregation pathway as well as early detection, which is the first step towards effective control of this key quality attribute • Protein aggregate/subvisible particle analysis should begin ‘upstream’ of drug product development (stable drug product begins with stable drug substance) • One can speed up drug product development and ensure long term sustainability by optimizing biologics formulation and integrating it with delivery device and analytical technology. • The increased regulatory and market expectation for high quality biopharmaceuticals creates opportunities for those who fully embrace drug product formulation, analytical, and drug delivery expertise. These are the backbone of every successful commercial product!
- 39. Thank you! Compassion BioSolution, LLC Danny K. Chou, PharmD, PhD E-mail: pharmd98@gmail.com Phone (USA): 303-483-3690 Compassionbiosolution.com