Automated Low Volume Subvisible Particle Analysis with the ... · Subvisible particle analysis is a...
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Automated Low Volume Subvisible Particle Analysis with the HORIZON System Bernardo Cordovez 1 ; Colby Ashcroft 1 ; Caitlin Wood 2 ; Renee Tobias 1 1 Halo Labs, 2 University of Delaware Department of Chemical and Biomolecular Engineering INTRODUCTION Subvisible particle analysis is a key predictor of protein drug stability and an essential drug product quality metric. However, current subvisible methods require large volumes of precious protein sample and are labor- and time-intensive, making it almost impossible to obtain this vital information during late stage candidate screening or early in formulation. The HORIZON ® system from Halo Labs employs Backgrounded Membrane Imaging (BMI) technology to measure and characterize subvisible particles in a high-throughput, low volume format. BMI is fully automated, fluidics- free, and uses only 1/10th the sample volume of conventional methods. Because particles are measured on a membrane surface and not in solution, BMI is also insensitive to solution refractive index, allowing it to reliably measure translucent protein aggregates. Samples are simply pipetted onto disposable 96-well membrane plates, vacuum filtered, then automated imaging software quickly generates information on counts, size, and distribution of particles captured on the membrane. Here we present data that demonstrates particle sizing and counting performance of the HORIZON ® system using certified USP standards with traceability to National Institute of Standards and Technology (NIST). CONCLUSION The HORIZON ® system, a subvisible particle analysis tool powered by its novel Backgrounded Membrane Imaging (BMI) technology enables: • Low volume: Requires as little as 25 μL of sample; • High throughput: Automated measurements enable completion of a full 96 well particle screening assay within 2 hours or 1 min per sample; • Wide working range: Measures particles from 2 µm to 4 mm with high reproduciblity (CVs <10% for polydisperse samples); • Measurement of samples with low Refractive Index contrast particles, such as protein aggregates; • Use of NIST-traceable standards for sizing and counting to verify performance. MORE SENSITIVE SUBVISIBLE PARTICLE ANALYSIS The HORIZON ® system produces data comparable to other subvisible systems including histograms and images. A comparison of Flow Imaging Microscopy with the HORIZON ® system is shown here. While trends are similar, the HORIZON ® counts more particles in each size bin (especially for translucent particles) due to the refractive index advantages of BMI. SIZING ACCURACY ON THE HORIZON ® SYSTEM The HORIZON ® system was used to measure diameter of four different sized NIST-traceable monodisperse polystyrene bead standards. Eight membrane wells (50 µL per well) were analyzed for each bead size, and average measured diameters and standard deviations are reported in the table below. Sizes were determined to within 2.2% of the bead specifications, demonstrating accuracy of measurement by BMI. COUNTING PRECISION ON THE HORIZON ® SYSTEM USING RM 8634 ETFE STANDARDS Reference Material (RM) 8634 is a NIST polydisperse particle standard made from abraded ethylene tetrafluoroethylene (ETFE). ETFE particles are similar in morphology and refractive index to protein aggregates. RM 8634 was measured on 96 wells of a HORIZON ® membrane plate (30 µL/well). Cumulative distribution curves were plotted from NIST reference values and HORIZON ® measurements (Chart A). While more particles are counted in each bin on the HORIZON ® system, the distribution curve shapes are similar. Higher counts can be attributed to platform-platform differences (flow system versus membrane surface measurement) and BMI’s ability to measure more translucent particles (higher refractive index contrast). Charts B and C demonstrate consistency of ETFE measurements on the HORIZON ® system. Combined variation in measured ETFE counts due to instrument, user, and sampling combined is less than 9%. HORIZON Particle Counts (particles/mL) FlowCam ® PV-100 Particle Counts (Particles/mL) Diameter Size Bin (µm) 0 5000 10000 Particle Counts (particles/mL) 10 5 15 20 15000 Flow Imager HORIZON ® Step 4 Data Visualization Step 3 Measurement and Analysis Step 1 Background Imaging Step 2 Sample Filtration 0 2,000 50 100 500 200 1,000 10 5 20 15 25 30 35 40 45 50 55 60 440 Particles/mL 220 Particles/mL 703 Particles/mL 1,275 Particles/mL 2066 Particles/mL 3,121 Particles/mL 4,176 Particles/mL 7,560 Particles/mL Particle Count (per mL) Diameter [ECD] (µm) Brightfield Intensity Diameter [ECD] (µm) 140 160 120 100 80 60 40 20 0 0 20 40 60 80 100 120 140 160 Background Measure Raw Measure BMI OVERVIEW MEMBRANE MICROSCOPY RE-IMAGINED Backgrounded Membrane Imaging (BMI) has its roots in USP <788>. In the USP method, samples are vacuumed through a filter plate and particles are captured on a flat membrane where they are manually counted by light microscopy. In BMI, this technique has been modernized using robotics, image processing, and innovative optics. First a background image of the membrane is taken, then samples are vacuumed through the filter and reimaged. The background and sample raw images are processed together in order to remove the background texture and clearly identify the particles present in the sample. NIST BEAD STANDARD (µm) DIAMETER (µm) DIAMETER STD DEVIATION HORIZON MEASURED ECD AVERAGE (µm) N=8 HORIZON MEASURED ECD STD DEVIATION (µm) N=8 5 5.01 0.04 4.9 0.06 10 10 0.08 10.0 0.15 15 15.02 0.15 15.0 0.12 30 29.75 0.56 29.4 0.28 NIST-traceable bead standards as measured by the HORIZON ® system. Cumulative distribution of RM 8634 NIST reference values versus the HORIZON ® system. Reproducibility of BMI measurements using NIST ETFE standard, 30 µL sample/2 µm cumulative bin. Chart B shows user–user variation (3 users, n=16 wells). Chart C shows instrument–instrument variation (2 instruments, n=24 wells). 0 10000 20000 30000 Counts/mL 0 5 10 15 20 25 Cumulative Size Bin HORIZON NIST A 0 10,000 20,000 30,000 Counts/mL User 1 User 2 User 3 B 0 10,000 20,000 30,000 Instrument 1 Instrument 2 Counts/mL C
Transcript of Automated Low Volume Subvisible Particle Analysis with the ... · Subvisible particle analysis is a...
Automated Low Volume Subvisible Particle Analysis with the HORIZON System
Bernardo Cordovez1; Colby Ashcroft1; Caitlin Wood2; Renee Tobias1 1 Halo Labs, 2 University of Delaware Department of Chemical and Biomolecular Engineering
INTRODUCTIONSubvisible particle analysis is a key predictor of protein drug stability and an essential drug product quality metric. However, cur rent subvisible methods require large volumes of precious protein sample and are labor- and time-intensive, making it almost impossible to obtain this vital information during late stage candidate screening or early in formulation. The HORIZON® system from Halo Labs employs Backgrounded Membrane Imaging (BMI) technology to mea sure and characterize subvisible particles in a high-throughput, low volume format. BMI is fully automated, fluidics-free, and uses only 1/10th the sample volume of conventional methods. Because particles are measured on a membrane surface and not in solution, BMI is also insensitive to solution refractive index, allow ing it to reliably measure translucent protein aggregates.
Samples are simply pipetted onto disposable 96-well membrane plates, vacuum filtered, then automated imaging software quickly generates information on counts, size, and distribution of particles captured on the membrane. Here we present data that demonstrates particle sizing and counting performance of the HORIZON® system using certified USP standards with traceability to National Institute of Standards and Technology (NIST).
CONCLUSION The HORIZON® system, a subvisible particle analysis tool powered by its novel Backgrounded Membrane Imaging (BMI) technology enables:
• Low volume: Requires as little as 25 μL of sample; • High throughput: Automated measurements enable completion of a full 96 well particle screening assay within 2 hours or 1 min per sample;
• Wide working range: Measures particles from 2 µm to 4 mm with high reproduciblity (CVs <10% for polydisperse samples);
• Measurement of samples with low Refractive Index contrast particles, such as protein aggregates; • Use of NIST-traceable standards for sizing and counting to verify performance.
MORE SENSITIVE SUBVISIBLE PARTICLE ANALYSISThe HORIZON® system produces data comparable to other subvisible systems including histograms and images. A comparison of Flow Imaging Microscopy with the HORIZON® system is shown here. While trends are similar, the HORIZON® counts more particles in each size bin (especially for translucent particles) due to the refractive index advantages of BMI.
SIZING ACCURACY ON THE HORIZON® SYSTEMThe HORIZON® system was used to measure diameter of four different sized NIST-traceable monodisperse polystyrene bead standards. Eight membrane wells (50 µL per well) were analyzed for each bead size, and average measured diameters and standard deviations are reported in the table below. Sizes were determined to within 2.2% of the bead specifications, demonstrating accuracy of measurement by BMI.
COUNTING PRECISION ON THE HORIZON® SYSTEM USING RM 8634 ETFE STANDARDSReference Material (RM) 8634 is a NIST polydisperse particle standard made from abraded ethylene tetrafluoroethylene (ETFE). ETFE particles are similar in morphology and refractive index to protein aggregates. RM 8634 was measured on 96 wells of a HORIZON® membrane plate (30 µL/well). Cumulative distribution curves were plotted from NIST reference values and HORIZON® measurements (Chart A). While more particles are counted in each bin on the HORIZON® system, the distribution curve shapes are similar. Higher counts can be attributed to platform-platform differences (flow system versus membrane surface measurement) and BMI’s ability to measure more translucent particles (higher refractive index contrast). Charts B and C demonstrate consistency of ETFE measurements on the HORIZON® system. Combined variation in measured ETFE counts due to instrument, user, and sampling combined is less than 9%.
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FlowCam® PV-100 Particle Counts (Particles/mL)Diameter Size Bin (µm)
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Step 2 Sample Filtration
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7,560 Particles/mL
5,670 Particles/mL
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BMI OVERVIEWMEMBRANE MICROSCOPY RE-IMAGINEDBackgrounded Membrane Imaging (BMI) has its roots in USP <788>. In the USP method, samples are vacuumed through a filter plate and particles are captured on a flat membrane where they are manually counted by light microscopy. In BMI, this technique has been modernized using robotics, image processing, and innovative optics. First a background image of the membrane is taken, then samples are vacuumed through the filter and reimaged. The background and sample raw images are processed together in order to remove the background texture and clearly identify the particles present in the sample.
NIST BEAD STANDARD (µm)
DIAMETER (µm)
DIAMETER STD DEVIATION
HORIZON MEASURED ECD AVERAGE (µm) N=8
HORIZON MEASURED ECD STD DEVIATION (µm) N=8
5 5.01 0.04 4.9 0.06
10 10 0.08 10.0 0.15
15 15.02 0.15 15.0 0.12
30 29.75 0.56 29.4 0.28
NIST-traceable bead standards as measured by the HORIZON® system.
Cumulative distribution of RM 8634 NIST reference values versus the HORIZON® system.
Reproducibility of BMI measurements using NIST ETFE standard, 30 µL sample/2 µm cumulative bin. Chart B shows user–user variation (3 users, n=16 wells). Chart C shows instrument–instrument variation (2 instruments, n=24 wells).
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