Medimmune_022108

MFI™ Flow Microscopy Technology

Feb 21, 2008

Martin Wang

Presentation Outline

• Particle Counting Techniques & Limitations• Flow Microscopy Technology• MFI™ Performance• MFI™ Applications• Conclusions • Questions/Discussion

WIS Biomed

Technical sales and distributorCurrent 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

Light Extinction/Obscuration

Measurement Principal • Pump Sample Through Cell• One Particle at a Time• Light Blockage Correlated to Particle Size

Strengths• Rapid & Objective• Low Concentrations• Well Established

“Quantification of Protein Particles in Parenteral Solutions using Light Obscuration and Micro Flow Imaging”Chi-Ting Huang; Immunogen Corporation Limitations

• Heterogeneous Particle Populations• Translucent Particles• Concentration Limit (10,000’s/ml)• Sensitivity to Air Bubbles• No Information Regarding Particle Origin

Manual Microscopic Analysis

Measurement Principal • Filter Sample (membrane filter, <1um)• Allow Filter to Dry• Count and Size Particles Using Reticule

Strengths• Particle Images• Heterogeneous Particles• High Magnification (~100X)

USP <788> Particulate Matter in Injections

Limitations• Labor Intensive• Operator Subjective• Translucent Particles (contrast)• Fragile Particles (damage)

Detection Zone

Flow Microscopy

Measurement Principal:• Sample Drawn Through Flow Cell• Fluid Imaged Digitally• Image Analysis

Strengths:• Count, Size, Shape• Heterogeneous Particles• Translucent Particles• Broad Concentration Range• Particle Classification

Limitations:• Small Particles (~nm)• Slightly Slower than Light Obscuration

Brightwell MFI Flow Microscopy Technology

Technology Comparison

Particle Classification

Fragile or Easily Deformed

HeterogeneousOptical Properties

ConcentrationMeasurement (#/ml)

Laser Diffraction

IrregularlyShaped Particles

Very Low Concentrations

Rapid ObjectiveAnalysis

Translucent or Near Transparent

Flow Microscopy

Manual Microscopic Analysis

Light Obscuration

Particle MeasurementChallenge

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)

Accuracy (DPA4100 Particle Analysis System)

Material Insensitivity (DPA4100 Particle Analysis System)

• Silica Microspheres (RI = 1.43), Source: Bangs Laboratories• Ethylene Glycol (RI=1.3335 to 1.3931 at 20°C from 0.5% to 60% by

mass in water)

Light Obscuration Material Sensitivity

Techniques for the Assessment of Droplet size in Parenteral Emulsions; Clive Washington; AstraZeneca

Contaminants:• Size and Count (#/ml) low concentrations of particles• Identify their origin by viewing images (indigenous vs. exogenous, air bubbles, etc…)• Quantify relative concentrations by applying filters on morphological parameters to isolate populations of

interest

Suspensions:• ECD Size distributions (number weighted or volume weighted)• Fiber length measurement (maximum Feret’s Diameter)• Other morphological parameter (circularity, aspect ratio…)• Relative concentrations of mixtures by applying filters on morphological parameters to isolate

populations of interest• Identification of particles causing unusual test results (e.g. air bubbles or small number of large particles)• Continuous monitoring (24 hrs) of sample and trend charting for dynamically changing particle

populations

MFI Applications

Formulation Stability(Protein Formulations)

Protein Formulation Particles

Particles in parenterals have traditionally been characterized by their source:• Packaging materials, manufacturing factors, formulation components,

miscellaneous sources • Major focus on contaminants (glass, silicone, metal, rubber, etc.)

Protein formulations present a new challenge to particle detection and measurement. Particles formed by protein aggregation are:• Highly transparent• Irregular in size and shape• Fragile and easily formed/deformed• Possibly present in very low concentrations (rare events)

Particles with these characteristics may not be reliably detected and measured with existing techniques

Increased Sensitivity to Protein Aggregates

• Protein aggregates are translucent, fragile, and highly irregular in shape• Light Obscuration and Manual Microscopy may not accurately measure some aggregates

European Journal of Parenteral & Pharmaceutical Sciences 2007; 12(4): 97-101

Formulation Stability

• Stability of formulation measured in terms of particle count as a function of time• Sensitive measurement of differences allowing formulation optimization

Quantification of Protein Particles in Parenteral Solutions using Light Obscuration and MFI_July'07_ImmunoGen Inc.

Image Comparison

Time (hr)0

2

4

6

24

24 hr, 0 rpm

PBS Buffer Formulation Improved Formulation


Formulation Filtration

• Pre- and post-0.22 µm filtration proteinaceous samples measured with obscuration and flow microscopy

• Similar relative size distributions, however MFI detects significantly more particles

• Images reveal the particles to be fluffy aggregates


Particle Classification(Protein Formulations)

Air Bubble Detection

• Coalescent air bubbles identified using intensity/aspect ratio filter• 98% of >3000 particles/ml identified as bubbles/bubble clusters


Single Bubble

CoalescedBubble

Protein Aggregate

Silicon Oil Droplet Detection

Silicone Oil Droplet

Protein Particle

• High particle counts in ≥10 and ≥25μm ranges using obscuration• Morphological Filters identify 61% of the total particle population as silicone oil droplets


Rubber, Metal, Glass

Protein Particle

Rubber Particle

Metal Particle

Glass Particle

Other Interesting Applications

Blood Cell Product Stability

Observations:• Cells become more spherical and shrink in diameter as they age• Maximum Feret’s Diameter found to be highly sensitive to these affects• Images confirm effects and correlate well with limited statistics available through manual microscopy

Blood Control

Blood Test Positive

Purification/HPLC Bead Uniformity

Observations:• Glass beads used for separation of blood sample constituents• Non-conforming beads can lead to false positives• Rapid direct measurement of percent non-conforming, plus images and morphology characterization

Whole Beads

Non-Conforming Beads

Cell Rupturing

Final Culture(Low Magnification)

Observations:• Protein producing yeast cells: before/after rupture• Size, concentration, transparency• Process control and pass optimization

After Rupture(Low Magnification)

MFI Customers Include:

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.

Thank You!

www.WISbiomed.com

www.Brightwelltech.com

Other Analysis Examples

Cerium Oxide Slurry Outliers

Observations:• MFI found Slurry A to possess consistently more particles/ml with increasing particle size• LPC identified Slurry A to possess more particles/ml <10µm, but fewer >10µm

MFI Particle Images

150µm50µm

Raw Water Particles

Settled Water Particle

Filtered Water Particles

150µm150µm

100µm

Observations:• Raw water, settled water, filter effluent• Concentration, size, selective image capture• Particle removal effectiveness, ‘nature’ of remaining particulates

WTP Particle Removal

Waste Water Sludge Biomass Formation

Observations:• Mixed culture (fungal vs. bacteria) wastewater biomass at pH 4.0 vs. pH 3.5 • Size, concentration, shape• Relative concentrations and sensitivity to environmental conditions

pH 3.5 150µm x 150µm

pH 4.0 450µm x 450µm

Coagulation/Flocculation Dynamics

Observations:• Kaolin clay with alum coagulant: impeller, G value, flocculation time, location within tank• Size and concentration vs. time, time stamped image capture• Optimization of coagulation/flocculation/sedimentation process

Cryptosporidium vs. Giardia Classification

Image Capture(50µm x 50µm)

Observations:• Cryptosporidium parvum and Giardia intestinalis• Mixed sample, stored in formalin• Size, concentration, selective image capture, shape (circularity)• Discrimination of microorganisms using size and shape

Ground Water Contamination

225 µm

600 µm

750 µm

175 µm

After Transfer Building

Observations:• Ground water particulate• Holding tower, after transfer building• Size, concentration, selective image capture• Identify source of biomass creating turbid water in distribution system

Surface Water Particulate

150 µm

150 µm

Selective Image Capture

Observations:• Lake water• Concentration, size, selective image capture• Determine appropriate form of treatment, study impact on UV disinfection

Blood Clot Detection

Blood Cell Aggregates >50µm

Observations:• Blood Sample• Concentration, size, selective image capture• Determine appropriate form of treatment, study impact on UV disinfection

RBC Degradation

Observations:• Whole blood stored in EDTA at RT and 4 ºC• Size & concentration vs. time• Monitor cell population changes between storage conditions over time

Cell Viability – Trypan Blue

Live Cell

Dead Cell

Observations:• HeLaT4+ (human cervical epithelial carcinoma)• Size, concentration, selective image capture, shape (circularity)• Relative concentration of agglomerated resin

Cell Lysing

Live Cells (150µm x 150µm)

Dead Cells(150µm x 150µm)

Observations:• MDCK cells lysed with ethanol• Size, concentration, transparency• Relative concentration of lysed cells

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.)

Observations:• Candida albicans cultured in Trypicase-Soy broth• Size, concentration, transparency, shape (circularity)• Classify yeast form vs. hyphal form

Toner Rheology

Representative Images(50µm x 50µm)

Observations:• Chemically produced toner diluted in water• Size, concentration, shape (circularity)• Control of size and shape (product performance)

Silica Adhesive Outliers

Representative Images(150µm x 150µm)

Observations:• Adhesive Silica Powder• Mixed with water• Size, concentration, selective image capture, shape (circularity)• Enumeration of non-conforming particles, agglomeration detection

Zeolite

Process Start Process Finish

Observations:• Zeolite petrochemical adsorbent• Sampled as function of time during fabrication• Size, concentration, transparency• Metrics for process control and cut-off

Salmon Semen

Representative Images

Observations:• Salmon semen• Diluted in Phosphate Buffered Saline • Size, concentration, selective image capture, shape, transparency• General characterization

Supplemental - Technical

Material Insensitivity (MFI DPA4100 Particle Analysis

System)

DPA4100 Specifications

System Configuration: Low Mag High Mag

• Optical Magnification 5X 14X• Field of View (FOV) 1,760 x 1,400 µm 620 x 500 µm• Flow Cell Depth 400 µm 100 µm

System Performance:

• Cell Size Range 2.25 to 400 µm 750 nm to 100µm• Sizing Resolution 0.25 µm 0.25 µm• Analysis Time (µL/min) 200 5.5• Analysis Time (particles/sec) 350 50• Concentration Limit ~275,000 ~800,000 (# 2.5 µm particles)

Image Characteristics:

• Pixel Resolution 1280 x1024 1280 x1024• Bit Depth 10 bit 10 bit• Pixel Area (5µm particle) ~75 ~300• Pixel Area (10µm particle) ~150 ~750

USP Reference Standard

Image Analysis & Data Presentation

MFI Sample Introduction

• Gravity-assisted for dense particles

• Drawing sample through flow cell minimizes particle fragmentation

• Syringe stirrer vs. magnetic stirrer minimizes particle fragmentation

• Low flow rates (<1ml/min) for fragile particles

• Variable stir rate for optimal particle dispersion

Depth of Field

0µm

50µm 100µm 150µm 200µm

Low Mag vs. High Mag

DPA4100 @ 4.9X (150µm x 150µm)

DPA4100 @ 13.8X (50µm x 50µm)

Representative Images

Material: Mulberry PollenSamples: Duke Scientific standard; 12-13µmAnalysis: Size, concentration, selective image acquisitionOutcome: General characterization

More MFI Customers:

Medimmune_022108

Health & Medicine

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