Standardization of Optical Particle Counters
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1 Standardization of Optical Particle Counters Dean Ripple Bioprocess Measurements Group NIST, Gaithersburg WCBP, January 25, 2012
2 Protein Particulates in Biotherapeutics Proteins in solution partially denature and subsequently agglomerate Highly hydrated ( 95% water) Some evidence of immunogenic properties Particulate size from 10s of nm to 100 µm 50µm Limitations of existing standards: No particles with low aspect ratios No highly irregular particles No particles with low optical contrast Existing standards have high density Current state-of-the-art Differing methods disagree by order of magnitude No means of standardizing instruments for response to protein particulates
3 Outline Drug reference standards vs. analytical standards for particles Available standards d Development of reference materials at NIST Discussion of two methods: light obscuration & flow microscopy Modeling light obscuration Using the discrepancy between microscopy and light obscuration to advantage Conclusions
4 Issues with Protein-Based Particle Standards Standards using actual proteins are limited by several factors: stabilization of protein particles likely requires storage and transport at 80 C, use of actual proteins precludes the use of accurate quantification techniques such as scanning electron microscopy protein particles are themselves quite variable, so there is no single protein that would match all applications Which morphology do we mimic? (variable scale)
5 Particles and Drug Reference Standard Drug Reference Standard: d For particle counting purposes, the reference standard may differ from actual product: Storage conditions generally differ (longer period frozen, shorter period on the shelf) Fill and finish may differ Reference standard not designed to have uniform particle load Proposal: Use drug-product reference standard to generate reference data: typical protein particle morphology for various product stresses particle size distribution for various well-controlled stresses (thermal exposure, agitation) ratio of light scattering cross section to actual cross section (more on this later) Calibrate counting instruments using analytical standards; compare test results on product with data from reference standard.
6 Invisibility of Particles with Low Optical Contrast Electrical sensing zone (Coulter) measurements give higher counts than flow microscopy Fluorescently labeled l protein aggregates visible ibl in larger numbers than with microscopy alone Refractive index measurements of particles give refractive index of particle as small as above fluid In focus Out of focus virtually invisible now visible digitally edge enhanced detection
7 Polystyrene latex (PSL) beads Available Analytic Standards good for calibration of concentration and diameter bracket size range of interest Monodisperse silica or polymethylmethacrylate (PMMA) beads lower refractive index, combined with higher refractive index liquid, better mimics protein particles not as readily available as PSL, methods not yet established Polydisperse glass bead standards good cross check on results from monodisperse PSL standards, to check for non-linearities over large size range requires care in method development so beads do not drop out of solution Gaps No good standards for irregular particle with with low optical contrast No good morphology standards
8 Abraded Polymer Particles at NIST Refractive Index Table Standards Materials Protein & Silicone Materials Polystyrene e Silicone oil 1.40 PMMA(acrylic) Adsorbed protein 1.40 Silica Protein solution (100 mg/ml) 1.35 to 1.36 ETFE (fluoropolymer) 1.40 Protein particles 1.34 to 1.40 PTFE (fluoropolymer) 1.35 Water ETFE polymer (tetrafluoroethlyene/ethylene copolymer) has desirable properties: 1. Refractive index of 1.40 ETFE 2. Very durable & tough 3. Can make it look like a protein diamond in a mechanical abrasion process motor
9 ETFE in water + 24 % sucrose (Δn = 0.03) ETFE in water (Δn = 0.07) Agitated IgG
10 Engineered Particles Borrow from semiconductor manufacturing technology: Fabricated polymer films (SU-8 photoresist) A single 150 mm wafer can produce 2x10 8 particles of area 40 µm 2 4 x 40 µm rods used to Potential size range from visible to < 1 µm study particle alignment in flow cells
11 Light Obscuration Particle passing through light beam reduces optical transmission Diffraction effects for small particles must be accounted for Scattering cross section depends on particle morphology & refractive index
12 Light Obscuration/Scattering Instrument measures integrated light scattering, but reports the results as diameter of polystyrene bead with equivalent scattering Detection efficiency varies with particle refractive index The main error arising in light obscuration is not undercounting, but undersizing
13 Instrument Response Model Approach: 1. Model particles as spheroids with no internal structure 2. Obtain the average refractive index of the particles from auxiliary measurements 3. Obtain the average aspect ratio from flow microscopy 4. Calculate the instrument response using mathematical light scattering models 5. Transform the LO data using the instrument response curve to estimate the actual particle diameter corresponding to the measured diameters. Transformation scales particle diameter, not count
14 Optical Method 2: Flow Microscopy Commonalities of instruments: t digital capture and analysis of particulates flat flow cells typical range of 1 to 100 µm Proprietary features: particle identification algorithm optical light source, camera, objectives, apertures Contrast t & spatial resolution depend d on proprietary choices
15 Results for Test with BSA
16 Results for Test with BSA
17 An Alternative Model works, but... correcting the light obscuration data is laborious requires measuring particle refractive index Alternative: Recognize that the discrepancy between light obscuration and flow microscopy data captures important particle information Let s use this discrepancy to advantage...
18 Diameter ratio
19 Finding the Scattering to Size Ratio Normalize the data 1. Start with counts per ml, N i, within bin i. 2. Create a new distribution M i from N i by dividing by Δd/d ave,where Δd is the bin width and d ave is the average diameter of the bin Advantages: M i doesn t depend on the choice of bin diameters, and larger bins can be used at larger particle sizes Find values of scattering diameter and image diameter that give the same counts 3. Find the values of d for flow microscopy and light obscuration that give the same values for M i 4. Compute the ratio of diameters
20 Diameter ratio, BSA particles
21 Monitoring Particle Counts Monitor counts from both light obscuration and flow microscopy to obtain: particle size distribution (historically what has been done) image diameter to scattering diameter ratio (new) Deviations in the diameter ratio indicate: changes in protein stress changes in levels l of contaminants t Analytic standards provide the primary traceability of instruments Drug reference standard contributes primarily through reference data for expected diameter ratio Future Work It may be possible to use reference standard more extensively if: particle counts in reference standard do not rise over lifetime particle generation methods can be highly reproducible
22 Analytic Standards d Conclusions NIST developing ETFE and lithographic standards: complex morphology & reduced optical contrast Method to correct light obscuration reduces 200x discrepancy to <10X. Lack of commercial method to measure particle refractive index Supplement common polystyrene y beads with silica or PMMA beads in matrix liquid of higher refractive index? Drug Reference Standard Reference data for typical protein particle morphology, optical contrast t of particles New Suggestion for Quality Control Light obscuration to flow microscopy diameter ratio gives empirical measure of optical contrast of particles
23 Acknowledgments Michael Carrier Dick Cavicchi Chip Montgomery Michael Tarlov Josh Wayment Rebecca Zangmeister
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