Rapid Particle Characterization of Therapeutic Protein Formulations Using

Continuous Digital Imaging

Lew Brown

Fluid Imaging Technologies, Inc.Yarmouth, ME USAwww.fluidimaging.com

Outline:

• Historical Usage of Microscope Images

• Limitations

• New Technologies for Analysis of Particulates (Benefits/Limitations)

• Fluid Imaging FlowCAM®: Architecture and Features

• Demonstration

Limitations of Traditional Microscopy

• Static Sample– Only a small amount of sample can be processed

at a time– Small sample = decreased reliability/repeatability

• Time to Prepare and Analyze Sample– Time required to prepare sample onto microscope

slide– Each particle counted and measured one at a time

• Human Error– “Tired eyes”– Interruptions

New Technologies for Analysis of Particulates (Benefits/Limitations)

• “Particle Analyzers”– Operate on “Particle Volume” Principle– Benefits:

• Non-static sampling• Very rapid counting

– Drawbacks:• “Particle Volume” only• No shape measurements• Limited information (“Relative Measurements”)• No picture!

New Technologies for Analysis of Particulates (Benefits/Limitations)

• Optical Automated Micrography– Originally photographic, now (mostly) digital– Benefits:

• “A picture is worth a thousand words!”• Automates measurements• Allows subjective review by scientist

– Drawbacks:• Static sample• Still time consuming

The Big Question:

FlowCAM Architecture

Continuous Imaging Particle Analysis: How it Works

Continuous Imaging Particle Analyzer: Features

• Continuous imaging• Fluorescence/scatter triggered imaging• Can collect upwards of 25,000 images/min• Intuitive software (including pattern

recognition) allows users to quickly navigate tens of thousands of images

–Qualify, isolate and quantify sub-populations based on any combination of the 23 measured parameters

–Remove unwanted particles from data sets

Analysis of Two Drug Formulations

• Goal–Provide a concentration of particles >10um

and >25um w/o counting the Si oil droplets that are common to these samples

–Provide images of these particles• Sample analysis

–10X Objective (~100X Overall Magnification), and a 90um deep flow cell

–Post-processed to remove Si oil droplets and air bubbles

Typical Results

Removing Unwanted Particles

• Display all particles >10um

• Use sorting or pattern matching to isolate unwanted images

• Delete unwanted images

• Display summary statistics for particles >10um and >25um

Effects of Processing• Before Droplet Removal

• After Droplet Removal

Summary of Results Sample A:

Summary of Results Sample B:

Formulations that contain high concentrations of particles larger than 10um have a higher chance of causing complications in patients than those that do not

Sample B has a many more outsized particles (492 p/ml) than sample A (95 p/ml)

Sample B is more apt to cause complications than than A

Conclusions

Flow Microscopy offers the Speed of Rapid P.A., With the Differentiation of

Microscopy

Speed enables the collection of statistically significant data populations

Instant visual feedback from particle images; great for outlier quantification and qualification

More data/particle provides an enhanced ability to identify and quantify different particle types

Flow Microscopy Offers the Speed of Rapid P.A., With the Differentiation of

Microscopy

All particle images saved; can be re-analyzed at any time, and can serve as concrete documentation

Morphological pattern matching allows for rapid data processing

Image libraries can be compiled and used as references for pattern matching (normalize out human error)

Thank You!

www.fluidimaging.com