ORTHOGONAL PARTICLE CHARACTERIZATION TECHNIQUES FOR BIO-APPLICATIONS: AN INTRODUCTION TO DLS (DYNAMIC LIGHT SCATTERING), NTA (NANOPARTICLE

Transcription

1 ORTHOGONAL PARTICLE CHARACTERIZATION TECHNIQUES FOR BIO-APPLICATIONS: AN INTRODUCTION TO DLS (DYNAMIC LIGHT SCATTERING), NTA (NANOPARTICLE TRACKING ANALYSIS) AND RMM (RESONANT MASS MEASUREMENT) - Dr. Markus Epe Field Application Scientist, Europe markus.epe@malvern.com

2 AGENDA Protein Aggregation Pathways and Subvisible Particles Technology Overview Orthogonal technologies Application Examples What is Resonant Mass Measurement (RMM) and where can I use it? 2 Title of the presentation 15 May 2018

3 PROTEIN AGGREGATION PATHWAYS AND SUBVISIBLE PARTICLES 3Title of the presentation 15 May 2018

4 Protein Aggregation SubVis / Vis Particles Adapted from: Chi et al., Pharm. Res. 20:1325 Protein Structure Hydrodynamic Size Particle Counts

5 Protein Aggregation SubVis / Vis Particles Adapted from: Chi et al., Pharm. Res. 20:1325 Protein Structure Hydrodynamic Size Particle Counts

6 PROTEIN AGGREGATES WHY SHOULD ANYONE CARE? Immunogenicity Aggregates may result in an unwanted immune response in the patient s body Loss of efficacy Other adverse effects Loss of efficacy Unfolded proteins do not have the same therapeutic effect Enumeration of particulates is REQUIRED by the US FDA and other regulatory agencies USP<787>, USP<788>, USP<790>

7 SUBVISIBLE PARTICLES Subvisible particle counts provide the most sensitive detection of protein aggregation. Formation of particles (nano & micro) is an early step on aggregation pathway. Even trace levels of particles can impact subsequent stability of protein solutions; Particles beget particles.

8 THE PARTICLE PROFILE Extrinsic Particle: not part of the formulation, packaging or assembly process. True contamination unexpected in drug product (hair, clothing fragments, insects, etc.) Intrinsic Particle: non protein particle that is associated with the container/closure (including delivery systems such as silicone oil), formulation components, manufacturing or packaging process. Inherent Particle: particles that originate from the drug product (protein therapeutic itself or formulation components). Narhi et al JPS

9 MANY FACTORS IMPACT PARTICLE PROFILES Manufacturing process Pumps Tubing Disposable technology Configuration Concentration Fill Volumes Container/closure/devices Formulation/excipients Shipping and handling Particle detection methods... The presence of visible particulate matter is one of the top 10 reasons for the recall of parenteral products Analysis of Aggregates and Particles in Protein Pharmaceuticals. Jiskoot and Mahler, 2012 So far in 2016, FDA has recalled 9 products for particular content

10 Orthogonal Technologies Particle Characterization Nanoparticles (1 nm 1 µm) SEC-MALS Taylor-Dispersion Analysis (Viscosizer TD) Nanosight (NTA) RMM (Archimedes) Subvisible Particles (1 µm 100 µm) Archimedes ( 6 µm) Light Obscuration (HIAC) Micro-Flow Imaging (MFI 5200) Raman Microscopy (Morphologi G4-ID) Visible Particles ( 100 µm) Visual Inspection Raman Microscopy Activities Validation of product specific methods Product/Process Characterization Biosimilarity/Comparability Stability testing Forced degradation testing Formulation testing Batch release testing Orthogonal USP <788>, USP <787> testing Raw Material ID Customized Image analysis Classification of particles (silicone oil droplets vs. protein, etc.) Raman chemical ID of particles (subvisible and visible)

11 Orthogonal Technologies Particle Characterization Nanoparticles (1 nm 1 µm) SEC-MALS Taylor-Dispersion Analysis (Viscosizer TD) Nanosight (NTA) RMM (Archimedes) Subvisible Particles (1 µm 100 µm) Archimedes ( 6 µm) Light Obscuration (HIAC) Micro-Flow Imaging (MFI 5200) Raman Microscopy (Morphologi G4-ID) Visible Particles ( 100 µm) Visual Inspection Raman Microscopy Activities Validation of product specific methods Product/Process Characterization Biosimilarity/Comparability Stability testing Forced degradation testing Formulation testing Batch release testing Orthogonal USP <788>, USP <787> testing Raw Material ID Customized Image analysis Classification of particles (silicone oil droplets vs. protein, etc.) Raman chemical ID of particles (subvisible and visible)

12 RESONANT MASS MEASUREMENT (ARCHIMEDES)

13 RMM what it does.. Weights particles in a fluid and differenciates the heavier from the lighter ones e.g. Protein, Nanomaterials from Oil or lipids Gives you size, weight and number of particles in solution RMM why using this.. DLS can give you size range but only qualitative information RMM offers Quantitative information about stability of proteins which is appreciated by authorities RMM helps to characterize proteins and define stability parametres To have a look into coating procedures of Nanomaterials Look into ultrafine bubbles (<1µm): recently published as having influence on stability for proteins in solution

14 OVERVIEW SIZE RANGES FOR ANALYTICS

15 Archimedes High Resolution SVP Characterisation Resonant Mass Measurement High resolution particle counts / concentration Protein aggregates: 200 nm 5 µm

16 Archimedes : Distinguishing Protein and Silicone By measuring buoyant mass, Archimedes can distinguish between particles are negatively buoyant, and those that are positively buoyant. Syringes and other containers commonly introduce oil droplets into therapeutic protein formulations Presence of oil droplets can compromise measurements of the quantity of protein aggregates

17 APPLICATION EXAMPLES

18 Archimedes: Characterising Protein Aggregates Assess impact of stress of SubVis particles Control Shear-stress Impact of shear stress on protein aggregates Obtain particle size distribution profile Compare different stress conditions Force Deg.

19 Case Study Comparison of IFN-β Products for Multiple Sclerosis 1994: Betaseron, IFN-β 1b (Bayer HealthCare) 1996: Lyophilized Avonex, IFN-β 1a (Biogen Idec) 2002: Rebif, IFN-β 1a (Merck Serono) - Liquid version of Avonex 2009: Extavia (Novartis), IFN-β 1b From: Barnard et al, J. Pharm. Sci. (2013): Vol.102,

20 ARCHIMEDES : DISTINGUISHING PROTEIN AND SILICONE Total Particles From: Barnard et al, J. Pharm. Sci. (2013): Vol.102,

21 Archimedes : Distinguishing Protein and Silicone Total Particles Protein Oil From: Barnard et al, J. Pharm. Sci. (2013): Vol.102,

22 ARCHIMEDES APPLICATIONS: COATING M. R. Nejadnik, W. Jiskoot Measurement of the Average Mass of Proteins Adsorbed to Nanoparticle by Using a Suspended Microchannel Resonator Pharmaceutics, Drug Delivery and Pharmaceutical Technology

23 RESONANT MASS MEASUREMENT: APPLICATION SUMMARY Counting particles analyzing mixtures of aggregates (50 nm 5 µm range) Determining total counts of proteinaggregates / particles and in a specific size range (200 nm-5µm) Determining surface coating on particles by weighting them: weight difference is due to coating applied Determining count depending on positive and negative buoyoncy: look into stabilization of solutions Looking into Nanobubble/ ultra fine bubble-applications: determine bubbles from other Materials in solution

24 SUMMARY ARCHIMEDES Gives quantitative data about protein aggregation in subvisible range (200 nm-5 µm for Proteins) and provides ortholog information to other technologies like Morphological Imaging and ID, MicroFlow Imaging, Light Obscuration, NTA in addition to qualitative information from DLS Small sample consumption ~100 µl Various applications: protein-aggregation, nanomaterials, coatings, milk skimming, stabilisation of emulsions / nanoparticle formulations etc. Quantifying protein aggregates and silicon oil by physical properties