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  1. 1. MFI™ Flow Microscopy Technology Feb 21, 2008 Martin Wang
  2. 2. Presentation Outline <ul><ul><li>Particle Counting Techniques & Limitations </li></ul></ul><ul><ul><li>Flow Microscopy Technology </li></ul></ul><ul><ul><li>MFI™ Performance </li></ul></ul><ul><ul><li>MFI™ Applications </li></ul></ul><ul><ul><li>Conclusions </li></ul></ul><ul><ul><li>Questions/Discussion </li></ul></ul>
  3. 3. WIS Biomed Technical sales and distributor Current products -NanoDrop technologies -Fujifilm QuickGene nucleic acid preparation -Biosensing Instrument: dual mode SPR molecule interaction detector -EVOS digital inverted microscope -Automated western, gel processor -24 sample homogenizer -Brightwell particle imager -Molecular biology reagents -Brady lab labeling and identification -Biotrue LIMS -Sundia Meditech CRO -TH lab chemical analysis -FMB antibody array services
  4. 4. Light Extinction/Obscuration <ul><li>Measurement Principal </li></ul><ul><ul><li>Pump Sample Through Cell </li></ul></ul><ul><ul><li>One Particle at a Time </li></ul></ul><ul><ul><li>Light Blockage Correlated to Particle Size </li></ul></ul><ul><li>Strengths </li></ul><ul><ul><li>Rapid & Objective </li></ul></ul><ul><ul><li>Low Concentrations </li></ul></ul><ul><ul><li>Well Established </li></ul></ul>“ Quantification of Protein Particles in Parenteral Solutions using Light Obscuration and Micro Flow Imaging ” Chi-Ting Huang; Immunogen Corporation <ul><li>Limitations </li></ul><ul><ul><li>Heterogeneous Particle Populations </li></ul></ul><ul><ul><li>Translucent Particles </li></ul></ul><ul><ul><li>Concentration Limit (10,000 ’ s/ml) ‏ </li></ul></ul><ul><ul><li>Sensitivity to Air Bubbles </li></ul></ul><ul><ul><li>No Information Regarding Particle Origin </li></ul></ul>
  5. 5. Manual Microscopic Analysis <ul><li>Measurement Principal </li></ul><ul><ul><li>Filter Sample (membrane filter, <1um) ‏ </li></ul></ul><ul><ul><li>Allow Filter to Dry </li></ul></ul><ul><ul><li>Count and Size Particles Using Reticule </li></ul></ul><ul><li>Strengths </li></ul><ul><ul><li>Particle Images </li></ul></ul><ul><ul><li>Heterogeneous Particles </li></ul></ul><ul><ul><li>High Magnification (~100X) ‏ </li></ul></ul>USP <788> Particulate Matter in Injections <ul><li>Limitations </li></ul><ul><ul><li>Labor Intensive </li></ul></ul><ul><ul><li>Operator Subjective </li></ul></ul><ul><ul><li>Translucent Particles (contrast) ‏ </li></ul></ul><ul><ul><li>Fragile Particles (damage) ‏ </li></ul></ul>
  6. 6. Flow Microscopy <ul><li>Measurement Principal: </li></ul><ul><ul><li>Sample Drawn Through Flow Cell </li></ul></ul><ul><ul><li>Fluid Imaged Digitally </li></ul></ul><ul><ul><li>Image Analysis </li></ul></ul><ul><li>Strengths: </li></ul><ul><ul><li>Count, Size, Shape </li></ul></ul><ul><ul><li>Heterogeneous Particles </li></ul></ul><ul><ul><li>Translucent Particles </li></ul></ul><ul><ul><li>Broad Concentration Range </li></ul></ul><ul><ul><li>Particle Classification </li></ul></ul><ul><li>Limitations: </li></ul><ul><ul><li>Small Particles (~nm) ‏ </li></ul></ul><ul><ul><li>Slightly Slower than Light Obscuration </li></ul></ul>Detection Zone Brightwell MFI Flow Microscopy Technology
  7. 7. Technology Comparison Particle Classification Fragile or Easily Deformed Heterogeneous Optical Properties Concentration Measurement (#/ml) ‏ Laser Diffraction Irregularly Shaped Particles Very Low Concentrations Rapid Objective Analysis Translucent or Near Transparent Flow Microscopy Manual Microscopic Analysis Light Obscuration Particle Measurement Challenge
  8. 8. DPA 4100 System Micro-Flow Imaging™ or MFI is Brightwell Technologies ’ flow microscopy technology MFI is designed to precisely control the volume represented by each image frame, and therefore permits accurate measurement of very low particle concentrations in addition to particle size and morphology. The DPA4100 is Brightwell ’ s R&D grade MFI system. Brightwell DPA 4100 System (pump and sample vessel not shown) ‏
  9. 9. Accuracy (DPA4100 Particle Analysis System) ‏
  10. 10. Material Insensitivity (DPA4100 Particle Analysis System) ‏ <ul><ul><li>Silica Microspheres (RI = 1.43), Source: Bangs Laboratories </li></ul></ul><ul><ul><li>Ethylene Glycol (RI=1.3335 to 1.3931 at 20°C from 0.5% to 60% by mass in water) ‏ </li></ul></ul>
  11. 11. Light Obscuration Material Sensitivity Techniques for the Assessment of Droplet size in Parenteral Emulsions; Clive Washington; AstraZeneca
  12. 12. <ul><li>Contaminants: </li></ul><ul><ul><li>Size and Count (#/ml) low concentrations of particles </li></ul></ul><ul><ul><li>Identify their origin by viewing images (indigenous vs. exogenous, air bubbles, etc … ) ‏ </li></ul></ul><ul><ul><li>Quantify relative concentrations by applying filters on morphological parameters to isolate populations of interest </li></ul></ul><ul><li>Suspensions: </li></ul><ul><ul><li>ECD Size distributions (number weighted or volume weighted) ‏ </li></ul></ul><ul><ul><li>Fiber length measurement (maximum Feret ’ s Diameter) ‏ </li></ul></ul><ul><ul><li>Other morphological parameter (circularity, aspect ratio … ) ‏ </li></ul></ul><ul><ul><li>Relative concentrations of mixtures by applying filters on morphological parameters to isolate populations of interest </li></ul></ul><ul><ul><li>Identification of particles causing unusual test results (e.g. air bubbles or small number of large particles) ‏ </li></ul></ul><ul><ul><li>Continuous monitoring (24 hrs) of sample and trend charting for dynamically changing particle populations </li></ul></ul>MFI Applications
  13. 13. Formulation Stability (Protein Formulations)
  14. 14. Protein Formulation Particles <ul><li>Particles in parenterals have traditionally been characterized by their source: </li></ul><ul><ul><li>Packaging materials, manufacturing factors, formulation components, miscellaneous sources </li></ul></ul><ul><ul><li>Major focus on contaminants (glass, silicone, metal, rubber, etc.) ‏ </li></ul></ul><ul><li>Protein formulations present a new challenge to particle detection and measurement. Particles formed by p rotein aggregation are: </li></ul><ul><ul><li>Highly transparent </li></ul></ul><ul><ul><li>Irregular in size and shape </li></ul></ul><ul><ul><li>Fragile and easily formed/deformed </li></ul></ul><ul><ul><li>Possibly present in very low concentrations (rare events) ‏ </li></ul></ul><ul><li>Particles with these characteristics may not be reliably detected and measured with existing techniques </li></ul>
  15. 15. Increased Sensitivity to Protein Aggregates <ul><ul><li>Protein aggregates are translucent, fragile, and highly irregular in shape </li></ul></ul><ul><ul><li>Light Obscuration and Manual Microscopy may not accurately measure some aggregates </li></ul></ul>European Journal of Parenteral & Pharmaceutical Sciences 2007; 12(4): 97-101
  16. 16. Formulation Stability <ul><ul><li>Stability of formulation measured in terms of particle count as a function of time </li></ul></ul><ul><ul><li>Sensitive measurement of differences allowing formulation optimization </li></ul></ul>Quantification of Protein Particles in Parenteral Solutions using Light Obscuration and MFI_July'07_ImmunoGen Inc.
  17. 17. Image Comparison Time (hr) ‏ 0 2 4 6 24 24 hr, 0 rpm PBS Buffer Formulation Improved Formulation Quantification of Protein Particles in Parenteral Solutions using Light Obscuration and MFI_July'07_ImmunoGen Inc.
  18. 18. Formulation Filtration <ul><ul><li>Pre- and post-0.22 µm filtration proteinaceous samples measured with obscuration and flow microscopy </li></ul></ul><ul><ul><li>Similar relative size distributions, however MFI detects significantly more particles </li></ul></ul><ul><ul><li>Images reveal the particles to be fluffy aggregates </li></ul></ul>Quantification of Protein Particles in Parenteral Solutions using Light Obscuration and MFI_July'07_ImmunoGen Inc.
  19. 19. Particle Classification (Protein Formulations)
  20. 20. Air Bubble Detection <ul><ul><li>Coalescent air bubbles identified using intensity/aspect ratio filter </li></ul></ul><ul><ul><li>98% of >3000 particles/ml identified as bubbles/bubble clusters </li></ul></ul>European Journal of Parenteral & Pharmaceutical Sciences 2007; 12(4): 97-101 Single Bubble Coalesced Bubble Protein Aggregate
  21. 21. Silicon Oil Droplet Detection Silicone Oil Droplet Protein Particle <ul><ul><li>High particle counts in ≥ 10 and ≥ 25 μ m ranges using obscuration </li></ul></ul><ul><ul><li>Morphological Filters identify 61% of the total particle population as silicone oil droplets </li></ul></ul>European Journal of Parenteral & Pharmaceutical Sciences 2007; 12(4): 97-101
  22. 22. Rubber, Metal, Glass Protein Particle Rubber Particle Metal Particle Glass Particle
  23. 23. Other Interesting Applications
  24. 24. Blood Cell Product Stability <ul><li>Observations: </li></ul><ul><ul><li>Cells become more spherical and shrink in diameter as they age </li></ul></ul><ul><ul><li>Maximum Feret ’ s Diameter found to be highly sensitive to these affects </li></ul></ul><ul><ul><li>Images confirm effects and correlate well with limited statistics available through manual microscopy </li></ul></ul>Blood Control Blood Test Positive
  25. 25. Purification/HPLC Bead Uniformity <ul><li>Observations: </li></ul><ul><ul><li>Glass beads used for separation of blood sample constituents </li></ul></ul><ul><ul><li>Non-conforming beads can lead to false positives </li></ul></ul><ul><ul><li>Rapid direct measurement of percent non-conforming, plus images and morphology characterization </li></ul></ul>Whole Beads Non-Conforming Beads
  26. 26. Cell Rupturing Final Culture (Low Magnification) ‏ <ul><li>Observations: </li></ul><ul><ul><li>Protein producing yeast cells: before/after rupture </li></ul></ul><ul><ul><li>Size, concentration, transparency </li></ul></ul><ul><ul><li>Process control and pass optimization </li></ul></ul>After Rupture (Low Magnification) ‏
  27. 27. MFI Customers Include:
  28. 28. Conclusions Flow microscopy combines the speed and convenience of obscuration counters with the visual insights offered by manual microscopic analysis. The higher sensitivity and material independence of flow microscopy improves particle detection and measurement of translucent, heterogeneous, and irregularly shaped particles. Morphology-based software filters provide rapid, accurate method for isolating and classifying particle sub-populations Breadth of measurement parameters and analysis features make the technology applicable to a large number of applications ranging from contamination detection to suspension characterization.
  29. 29. Thank You!
  30. 30. Other Analysis Examples
  31. 31. Cerium Oxide Slurry Outliers <ul><li>Observations: </li></ul><ul><ul><li>MFI found Slurry A to possess consistently more particles/ml with increasing particle size </li></ul></ul><ul><ul><li>LPC identified Slurry A to possess more particles/ml <10µm, but fewer >10µm </li></ul></ul>MFI Particle Images
  32. 32. WTP Particle Removal 150µm 50µm Raw Water Particles Settled Water Particle Filtered Water Particles 150µm 150µm 100µm <ul><li>Observations: </li></ul><ul><ul><li>Raw water, settled water, filter effluent </li></ul></ul><ul><ul><li>Concentration, size, selective image capture </li></ul></ul><ul><ul><li>Particle removal effectiveness, ‘ nature ’ of remaining particulates </li></ul></ul>
  33. 33. Waste Water Sludge Biomass Formation <ul><li>Observations: </li></ul><ul><ul><li>Mixed culture (fungal vs. bacteria) wastewater biomass at pH 4.0 vs. pH 3.5 </li></ul></ul><ul><ul><li>Size, concentration, shape </li></ul></ul><ul><ul><li>Relative concentrations and sensitivity to environmental conditions </li></ul></ul>pH 3.5 150µm x 150µm pH 4.0 450µm x 450µm
  34. 34. Coagulation/Flocculation Dynamics <ul><li>Observations: </li></ul><ul><ul><li>Kaolin clay with alum coagulant: impeller, G value, flocculation time, location within tank </li></ul></ul><ul><ul><li>Size and concentration vs. time, time stamped image capture </li></ul></ul><ul><ul><li>Optimization of coagulation/flocculation/sedimentation process </li></ul></ul>
  35. 35. Cryptosporidium vs. Giardia Classification Image Capture (50µm x 50µm) ‏ <ul><li>Observations: </li></ul><ul><ul><li>Cryptosporidium parvum and Giardia intestinalis </li></ul></ul><ul><ul><li>Mixed sample, stored in formalin </li></ul></ul><ul><ul><li>Size, concentration, selective image capture, shape (circularity) ‏ </li></ul></ul><ul><ul><li>Discrimination of microorganisms using size and shape </li></ul></ul>
  36. 36. Ground Water Contamination 225 µm 600 µm 750 µm 175 µm After Transfer Building <ul><li>Observations: </li></ul><ul><ul><li>Ground water particulate </li></ul></ul><ul><ul><li>Holding tower, after transfer building </li></ul></ul><ul><ul><li>Size, concentration, selective image capture </li></ul></ul><ul><ul><li>Identify source of biomass creating turbid water in distribution system </li></ul></ul>
  37. 37. Surface Water Particulate 150 µm 150 µm Selective Image Capture <ul><li>Observations: </li></ul><ul><ul><li>Lake water </li></ul></ul><ul><ul><li>Concentration, size, selective image capture </li></ul></ul><ul><ul><li>Determine appropriate form of treatment, study impact on UV disinfection </li></ul></ul>
  38. 38. Blood Clot Detection Blood Cell Aggregates >50µm <ul><li>Observations: </li></ul><ul><ul><li>Blood Sample </li></ul></ul><ul><ul><li>Concentration, size, selective image capture </li></ul></ul><ul><ul><li>Determine appropriate form of treatment, study impact on UV disinfection </li></ul></ul>
  39. 39. RBC Degradation <ul><li>Observations: </li></ul><ul><ul><li>Whole blood stored in EDTA at RT and 4 ºC </li></ul></ul><ul><ul><li>Size & concentration vs. time </li></ul></ul><ul><ul><li>Monitor cell population changes between storage conditions over time </li></ul></ul>
  40. 40. Cell Viability – Trypan Blue Live Cell Dead Cell <ul><li>Observations: </li></ul><ul><ul><li>HeLaT4+ (human cervical epithelial carcinoma) ‏ </li></ul></ul><ul><ul><li>Size, concentration, selective image capture, shape (circularity) ‏ </li></ul></ul><ul><ul><li>Relative concentration of agglomerated resin </li></ul></ul>
  41. 41. Cell Lysing Live Cells (150µm x 150µm) ‏ Dead Cells (150µm x 150µm) ‏ <ul><li>Observations: </li></ul><ul><ul><li>MDCK cells lysed with ethanol </li></ul></ul><ul><ul><li>Size, concentration, transparency </li></ul></ul><ul><ul><li>Relative concentration of lysed cells </li></ul></ul>
  42. 42. Yeast Cell Morphotypes (Images above are typical images in the following size ranges 1-2µm, 2-3µm, 3-4µm, 4-5µm, 6-7µm, 7-8µm, 8-10µm, 10-15µm, and >15µm respectively.) ‏ <ul><li>Observations: </li></ul><ul><ul><li>Candida albicans cultured in Trypicase-Soy broth </li></ul></ul><ul><ul><li>Size, concentration, transparency, shape (circularity) ‏ </li></ul></ul><ul><ul><li>Classify yeast form vs. hyphal form </li></ul></ul>
  43. 43. Toner Rheology Representative Images (50µm x 50µm) ‏ <ul><li>Observations: </li></ul><ul><ul><li>Chemically produced toner diluted in water </li></ul></ul><ul><ul><li>Size, concentration, shape (circularity) ‏ </li></ul></ul><ul><ul><li>Control of size and shape (product performance) ‏ </li></ul></ul>
  44. 44. Silica Adhesive Outliers Representative Images (150µm x 150µm) ‏ <ul><li>Observations: </li></ul><ul><ul><li>Adhesive Silica Powder </li></ul></ul><ul><ul><li>Mixed with water </li></ul></ul><ul><ul><li>Size, concentration, selective image capture, shape (circularity) ‏ </li></ul></ul><ul><ul><li>Enumeration of non-conforming particles, agglomeration detection </li></ul></ul>
  45. 45. Zeolite Process Start Process Finish <ul><li>Observations: </li></ul><ul><ul><li>Zeolite petrochemical adsorbent </li></ul></ul><ul><ul><li>Sampled as function of time during fabrication </li></ul></ul><ul><ul><li>Size, concentration, transparency </li></ul></ul><ul><ul><li>Metrics for process control and cut-off </li></ul></ul>
  46. 46. Salmon Semen Representative Images <ul><li>Observations: </li></ul><ul><ul><li>Salmon semen </li></ul></ul><ul><ul><li>Diluted in Phosphate Buffered Saline </li></ul></ul><ul><ul><li>Size, concentration, selective image capture, shape, transparency </li></ul></ul><ul><ul><li>General characterization </li></ul></ul>
  47. 47. Supplemental - Technical
  48. 48. Material Insensitivity (MFI DPA4100 Particle Analysis System) ‏
  49. 49. DPA4100 Specifications <ul><li>System Configuration : Low Mag High Mag </li></ul><ul><ul><li>Optical Magnification 5X 14X </li></ul></ul><ul><ul><li>Field of View (FOV) 1,760 x 1,400 µm 620 x 500 µm </li></ul></ul><ul><ul><li>Flow Cell Depth 400 µm 100 µm </li></ul></ul><ul><li>System Performance : </li></ul><ul><ul><li>Cell Size Range 2.25 to 400 µm 750 nm to 100µm </li></ul></ul><ul><ul><li>Sizing Resolution 0.25 µm 0.25 µm </li></ul></ul><ul><ul><li>Analysis Time (µL/min) 200 5.5 </li></ul></ul><ul><ul><li>Analysis Time (particles/sec) 350 50 </li></ul></ul><ul><ul><li>Concentration Limit ~275,000 ~800,000 </li></ul></ul><ul><ul><li>(# 2.5 µm particles) </li></ul></ul><ul><li>Image Characteristics : </li></ul><ul><ul><li>Pixel Resolution 1280 x1024 1280 x1024 </li></ul></ul><ul><ul><li>Bit Depth 10 bit 10 bit </li></ul></ul><ul><ul><li>Pixel Area (5µm particle) ~75 ~300 </li></ul></ul><ul><ul><li>Pixel Area (10µm particle) ~150 ~750 </li></ul></ul>
  50. 50. USP Reference Standard
  51. 51. Image Analysis & Data Presentation
  52. 52. MFI Sample Introduction <ul><ul><li>Gravity-assisted for dense particles </li></ul></ul><ul><ul><li>Drawing sample through flow cell minimizes particle fragmentation </li></ul></ul><ul><ul><li>Syringe stirrer vs. magnetic stirrer minimizes particle fragmentation </li></ul></ul><ul><ul><li>Low flow rates (<1ml/min) for fragile particles </li></ul></ul><ul><ul><li>Variable stir rate for optimal particle dispersion </li></ul></ul>
  53. 53. Depth of Field 0µm 50µm 100µm 150µm 200µm
  54. 54. Low Mag vs. High Mag DPA4100 @ 4.9X (150µm x 150µm) ‏ DPA4100 @ 13.8X (50µm x 50µm) ‏ Representative Images Material: Mulberry Pollen Samples: Duke Scientific standard; 12-13µm Analysis: Size, concentration, selective image acquisition Outcome: General characterization
  55. 55. More MFI Customers: