Page 41 - Pharmaceutical- Guide to Biopharmaceutical

Characterization Quality Control

Evaluating Aggregates in Protein Drug Products iSpect DIA-10

Characterization of Insoluble Subvisible Particles in benefits

Biopharmaceuticals Using the Flow Imaging Method Cell Line Optimization
click here • The optical system, which overlooks very few particles, can be used to evaluate the
particle count concentration of micrometer-level aggregates.
Operating Principle and Features Table 2 Observed Particle Count and Number Concentration
Heat aggregate sample Stirring aggregate sample • Sample quantities as small as 50 μL can be measured.
For protein drug evaluation, the United States Pharmacopeia and Overall 32246 18813 • Simple measurements can be performed in three steps, which minimizes the burden
Japanese Pharmacopoeia specify using the light obscuration (LO) Observed particle < 2 µm< 20129 4669
method to evaluate insoluble particles that are 10 μm or larger. count By size 2 - 10 µm 11797 14057 on operators. Culture
Meanwhile, flow imaging (FI), a dynamic image analysis method that 10 - 25 µm 298 78 9
≥ 25 µm
22
offers high sensitivity for particles with high permeability and also the Overall 668102 389784
ability to classify particles in images, has been gaining attention as a < 2 µm 417051 96737
method for analyzing micrometer-level insoluble subvisible particles. Particle 2 - 10 µm 244421 291246
concentration*
The iSpect DIA-10 dynamic particle image analysis system is used to (count/mL) By size 10 - 25 µm 6174 1616
acquire images of particles in liquid samples for measuring the size ≥ 25 µm 456 186
distribution, concentration, and shapes of particles based on the flow * Particle concentration is calculated from the observed particle count, volume of area observed,
imaging method. Due to the small sample quantities used (minimum 50 μL and number of images
for measurements) and the optical system that misses very few particles,
it is ideal for evaluating insoluble subvisible particles in biopharmaceuticals. Purification
Measurement Method and Conditions

Sample solutions were prepared using freeze-dried human
immunoglobulin. The sample powder was dissolved in a pH 5.0 citrate-
phosphate buffer solution (1 mg/mL), which was filtered through a 100
nm filter and the filtrate was used as the sample solution.
Half the sample solution was heated for three minutes at 80 °C.
The other half was stirred with a PEEK polymer stirring plate for ten
minutes. Then the heat-aggregated and the stirring-aggregated
samples were measured according to the conditions in Table 1. Characterization
Table 1 Measurement Conditions Specifications
Fig. 2 Particle Image of Aggregates from Stirring
Frame rate 8 frame / sec
Efficiency 97 % Instrument iSpect DIA-10
Heat stress
Sample amount 50 µL Stirring stress Measurement method Dynamic image analysis method
Threshold 220
Flowrate 0.1 mL / min Particle size measurement range *1 *2 5 to 100 μm
Results Concentration (count/mL) Particle count concentration reproducibility CV ≤ 5 % Quality Control
Measurement parameters
Particle size
The particle size distributions and scatter plots obtained from the Area circle equivalent diameter, perimeter equivalent diameter, maximum length,
maximum perpendicular length, vertical Feret diameter, horizontal Feret diameter,
measurements are shown in Fig. 1. Particle images are shown in Fig. 2. particle perimeter, envelope perimeter, etc.
The particle images can be used to distinguish between aggregates, air Shape analysis
bubbles, and oil droplets. Particle concentration measurement results All Circularity, aspect ratio, horizontal bounding rectangle aspect ratio, etc.
are shown in Table 2 and Fig. 3. Fig. 3 Count Concentration for Respective Particle Sizes Other parameters
Though it is difficult to analyze the shape of particles smaller than 5 Particle area, average brightness, etc.
μm, detecting them is possible. Conclusion Statistical analysis items Average, standard deviation, CV, median (50 % value), mode value, user defined %
iSpect DIA-10 systems can measure very small quantities of samples value
Absolute Amount (Diff)(count) Absolute Amount (Diff)(count) with excellent imaging efficiency. Even the type of insoluble subvisible Display items Particle image, histogram, scattergram, cumulative distribution, Pharmacokinetics
frequency/integration table, user defined area particle count
particles can be predicted from particle images, making it ideally
suited for evaluating the concentration of micrometer-level insoluble
Required sample volume
50 to 1000 μL
Area Based Diameter (µm) Area Based Diameter (µm) subvisible particles contained in biopharmaceuticals. Pump Syringe pump with 0.1 mL/min flowrate
Application Examples (Shimadzu Application News No.) Wetted part materials Measurement unit: PEEK resin, fluorine resin, quartz, or fluorine rubber
Aspect Ratio Aspect Ratio • Evaluating the concentration of insoluble particles in biopharmaceuticals Pump unit: Fluorine resin or glass

• Evaluating contaminants, coarse particles, and particle shapes in Dimensions and weight Measurement unit: W 223 mm × D 465 mm × H 205 mm, 10 kg Others
Area Based Diameter (µm) Area Based Diameter (µm) pharmaceuticals Pump unit: W 97 mm × D 190 mm × H 150 mm, 3 kg
a) Heat Aggregate Sample b) Stirring Aggregate Sample
• Evaluating the size of suspended particles in eye drops (Q122)
Fig. 1 Particle Size Distribution and Scatter Diagrams *1 Performance guarantee range of area circle equivalent diameter. Measured using a Shimadzu NIST traceable particle size standard sample.
*2 Measured using concentration standard samples specified by Shimadzu.

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