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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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