Peer Review Article | Open Access | Published 16th July 2024
Particle Matter determination in Biosimilar Parenteral Product by the Application of Dynamic Light Scattering (DLS) Followed by Statistical Evaluation
Akhilesh Kumar Kuril¹*, K Saravanan¹ | EJPPS | 292 (2024) | https://doi.org/10.37521/ejpps.29201 | Click to download pdf
Abstract
Particulate matter in parenteral dosage forms can emerge from numerous causes, external, intrinsic, as well as inherent within the product, with a special emphasis on biopharmaceuticals. Aqueous impurities, pharmaceutical precipitates, dirt, glass, rubber, pollutants from the environment, fibres, and various other insoluble materials are all common sources of particulates. When assessing the possible harm to patients, particulate matter size is a crucial issue to consider. Particles as fine as 2 μm overall diameter were found related with microthrombi development. The DLS (Dynamic Light Scattering) technique has been used to measure and control the subvisible particulate particles in biopharmaceutical parenteral drug products since the technique can measure the submicron particle size in the parenteral formulation. The purpose of using DLS is to measure and control the subvisible particles in a biopharmaceutical formulation. A generic biopharmaceutical product viz. Calcitonin Salmon injection was used for particulate matter analysis by using Dynamic Light Scattering.
DLS is a non-invasive method for detecting the size of suspended particles as well as molecules which is used for the control and optimization of processes, and the improvement of product quality and performance by analysing the time-dependence in regard to intensity of the dispersed light (auto correlation) to determine the diffusion speed (Brownian motion) of particles/molecules, and subsequently determine the hydrodynamic size.
Keywords: Particulate matter; DLS; Biosimilars; Parenteral dosage forms
1.Introduction
Particulate matter, whether visible as well as subvisible, especially sterile parenteral medications, is recognized as a key quality trait that impacts the product's safety¹. Particulate matter throughout injections as well as parenteral treatments consists of movable undissolved particles, in addition to gas bubbles, which are accidentally found within solutions². Particulate substance, either visible or subvisible, in a sterilized injectable dosage form is recognized as an important quality feature that affects safety and efficacy of the product resulting in an impact on patient health³. Particulate matter in parenteral a dosage form can emerge from numerous causes, external, intrinsic, as well as inherent towards the product, with a special emphasis on biopharmaceuticals⁴. Common particle sources include solvent contaminates, drug precipitation, dirt, transparent material, rubber, environmental pollutants, fibres, and various other insoluble substances⁵.
The size of particulate matter becomes a significant consideration when assessing the possible harm to patients². Particles as fine as 2 μm were discovered to be linked to microthrombi production in patients⁶. According to Dr. Michael Akers who works for the Food and Drug Administration (FDA), the finest blood capillaries measure around 7 μm in dimension. Particles larger than 7 μm can clog capillaries, increasing the risk of severe outcomes⁷. Simple visual evaluation, as necessary with compounding injections, might be sufficient for big particles but not for fine particles. About 40 μm is the limit of sight for tiny particles to the unaided eye. Specialized testing procedures are thus required to fully determine the entire particle load of injections².
The DLS technique has been used to measure and control the subvisible particulate particles in biopharmaceutical parenteral products since the technique can measure the submicron particle size in the parenteral formulation⁴. Dynamic Light Scattering (also known as Photon Correlation Spectroscopy and Quasi-Elastic Light Scattering) provides a method for detecting the size of particles, often in the submicron area⁷.
A generic biopharmaceutical product viz. Calcitonin Salmon injection was used for the Particulate matter analysis by using the DLS method.
DLS detects Brownian motion and correlates it with particle size. Brownian motion is the random rotation of particles caused through the bombardment generated by the solvent molecules around particles⁸. The larger the particle, the smaller the motion and vice versa. The speed of Brownian motion is affected by particle size, sample viscosity, as well as temperature⁹.
The velocity of the Brownian motion is defined by a property known as the translational diffusion coefficient (usually given the symbol D). The size of a particle is calculated from the translational diffusion coefficient by using the Stokes-Einstein equation
DLS is a non-invasive approach for determining the size of small particles as well as molecules under suspension which is used for the control and optimization of processes, and the improvement of product quality and performance by analysing the time-dependence in terms of intensity of the scattered light (auto correlation) to determine the diffusion speed (Brownian motion) of particles/molecules, and subsequently determine the hydrodynamic size¹⁰.
The six primary parts of a typical dynamic light scattering system are as follows.
In order to illuminate the sample inside a cell, a laser first supplies a light source⁷. At diluted concentrations, the majority of the laser beam goes through the sample, but some is dispersed in all directions by the particles in the sample. The dispersed light is measured using a detector. Depending on the model, the detector position in the Zetasizer Nano series will be at either 173° (backscatter detection angle), 90° (side scatter detection angle), or 13° (forward scatter), or a mix of the two⁷. For the detector to assess scattered light intensity properly, it has to fall into a certain range. The detector will get saturated if it detects too much light. In order to get around this, the intensity of the laser source, and consequently the intensity of scattering, are decreased using an attenuator. For samples that scatter little light, such as very small particles or materials with low concentrations, the amount of scattered light needs to be increased. The attenuator in this case will let more laser light reach the sample¹¹.
The detector's scattering intensity signal is sent to a correlator, a type of digital processing board. The rate at which the intensity is changing is determined by the correlator by comparing the scattering intensity at successive time intervals¹². After that, the computer receives this correlator data, which is processed by software to determine the size information.
In general, the main outcomes of DLS are the polydispersity index (PDI), which describes the distribution width of particle size, and the mean value of the intensity distribution, also known as the Z average¹¹.
To interpret the size data from the sample we must understand the following terminology from the software to obtain good quality data.
Count rate
The quantity of photons detected in a second is known as the count rate. In the real-time measurement display, count rate is presented. A sample may not be appropriate for DLS measurement if the count rate is less than 20 kcps, or if the count rate is more than 1000 kcps and the sample contains dust, aggregates, or large particles.
2. Measurement and attenuator positions
The optimum measurement position to obtain good quality data is 4.65 (Figure 2). In order to lower the strength of the laser source and, consequently, the intensity of scattering, an attenuator is utilized¹⁴. The software determines the proper attenuator position automatically, covering a transmission range of 100% to 0.0003%. The maximum attenuation (least laser power on the sample) is attenuator position 1, and the lowest attenuation (most laser power on the sample) is attenuator position 11¹⁵.
3. Correlator
A correlator is a type of digital processing board that receives the signal from the detector. We can also comprehend the sample more easily with the help of Correlogram’s form. (Figure 2)¹¹.
4. Z-Average Diameter (ZD)
The collection of particles' intensity-weighted mean hydrodynamic size (nm), as determined by dynamic light scattering (DLS). A study of the measured correlation function using Cumulants yields the Z-average¹⁶,¹⁷. It is the main and most reliable parameter that the method generates. According to ISO 13321, the Z-Average mean is the ideal number to report when utilized in a quality control situation.
5. Polydispersity index (PDI)
A dimensionless number that characterizes the distribution of particle sizes in a sample is produced by Cumulants analysis¹⁸. International standards organizations (ISOs) have determined that PI values > 0.7 are typical of a broad size (e.g., polydisperse) distribution of particles, whereas values < 0.05 are more common to monodispersed samples, which are rarely seen other than with highly monodisperse standards¹⁴,¹⁷. A value greater than 0.7 suggests that the sample is likely unsuitable for the DLS approach due to its wide size dispersion¹⁴,¹⁷.
A synthesized peptide variant of calcitonin called salmon calcitonin is used to treat hypercalcemia, osteoporosis, and Paget's disease by preventing bone resorption¹⁹. Salmon calcitonin is an alpha-helical polypeptide with 32 amino acids that is substantially different from human calcitonin from amino acids 10 to 27. The higher potency of calcitonin generated from salmon is explained by these variations in amino acid sequence. It functions by binding to a calcitonin receptor that is G protein coupled and mostly transduces signals through the PLC/IP3 and cAMP pathways²⁰. The physiologic effects of calcitonin on osteoclasts and the kidney's tubular epithelium are the most significant clinically. It lowers serum calcium and phosphate in the tubules by encouraging diuresis and inhibiting reabsorption. It makes osteoclasts in bone contract, lowering their motility and capacity to reabsorb bone²¹.
The Size of Protein and other biological molecules are presented in Figure 3
Materials and Methods
Materials
The sterile solution of calcitonin injection comes in volumes of 200 international units per mL or 400 international units per two mL vial. It is preserved with 0.5% phenol USP. To control tonicity, sodium chloride, sodium acetate, acetic acid, and sodium hydroxide have been added. Malvern Zetasizer: New series Zetasizer Ultra with ZS XPLORER software for Calcitonin Salmon injection at the application lab. Zetasizer advance series Ultra model with high power laser (10 mv) helps detecting below 5 nm particles, even if they have weak scattering ability and helps us to characterize proteins/polymers even detecting monomer fragments along with minor aggregates present, which can help in protein screenings.
Sample Preparation for Size Analysis
The sample was transferred from the sample vial into a measuring cuvette with the help of a 1 mL syringe. Precaution must be taken to avoid any bubble formation during transfer. About 1 mL of the sample is required for the size analysis, since the technique is non-invasive the same sample can be recovered from the cuvette to perform the other characterization tests. Glass cuvettes (PCS1115) were used for sizing analysis with as such sample without dilutions or filtration. Dispersant used is water. Viscosity, RI and Dielectric constant of water were 0.887 cP, 1.33 and 78.5, respectively for sample measurement. Size analysis was performed at 25 °C. Sample was equilibrated in the cuvette holder for 60 seconds inside the cuvette. The measurement was done at the forward scatter (13°), but the results showed an unreliable size distribution pattern. Following that the measurement angle kept was Backscatter (173°) and measurement time was kept in auto mode. A total of five measurements were taken and data processing was done in the general-purpose normal resolution mode. The results were reported by taking the average of five runs. Reproducibility (method precision) of the method was assessed by performing the above analysis six consecutive times. Interday variability (intermediate precision) was also assessed by analysing the sample in the above-mentioned conditions six times on different days. Additionally, the suitability of the method was performed by the statistical evaluation of the data using Minitab software.
Remark: Entire Precision study data was used for the statistical evaluation. To determine the method stability and capability, concept of Statistical Process Control (SPC) and Capability Six pack analysis with Upper Specification Limit (USL) 5 nm was applied to the data presented in Table 1 and Table 2 using Minitab.
Results and discussion
SPC was developed by Shewart in 1924. SPC is a method to operate a process efficiently using statistical methods. It uses statistical analysis tools to identify sources of variation and controls the process to meet the goals by applying a PDCA (Plan Do Check Action Plan) cycle (22) as per Walter Shewart. SPC is an outcome of an experiment that will be said to be controlled when, through the use of past experience, we can predict, at least within limits, how the phenomenon will behave in the future. SPC uses control charts to monitor the process. Since the particle size data is the continuous type of data and follows the normal distribution with subgroup size 1 hence the Individual Moving Range (I-MR) chart will be applied. An individual moving range (I-MR) chart was used to define the process stability and Six pack analysis was performed to determine the capability of process. Reproducibility results of Size analysis (PM) for Calcitonin Salmon injection are given in Table 1. Intermediate precision (Interday variability) of Size analysis (PM) for Calcitonin Salmon injection is given Table 2.
The overlaid size distribution curve from Precision study of Calcitonin Salmon injection is depicted in Figures 4 and 5 by Malvern Zeta sizer Ultra representing Intensity based distribution, Number based distribution and Correlogram from the study.
Figure 5: Overlaid particle size distribution curve from Intermediate Precision study; A: Intensity distribution, B: Number distribution, C: Correlogram by Malvern Zetasizer ultra
Control Charts indicate Special Causes of variation being either assignable causes or patterns and to determine the special cause the following Nelson tests were applied in the Minitab presented in Figure 6.
Figure 6: Tests to identify in Minitab to detect special cause
The results of the tests are presented in the Figures nos. 7-10.
Figure 7: I-MR chart for Particle size (Z avg.) from Precision study
Figure 8: I-MR chart for PDI evaluation from Precision study
Figure 9: Process capability six pack report for Z avg. from Precision study
Figure 10: Process capability six pack report for PDI from precision study
Z average and polydispersity index (PDI) are two index parameter which can address the particle size distribution in the sample and this information can be used for the assessment of particulate matter at the submicron level. Other measurement parameters such as mean count rate (kcps), measurement position, intercept and Attenuator will also be discussed under this section to understand instrument mechanism (Table 3).
Table 3: Results of other quality attributes in DLS for Size measurement
From the results mentioned in Table 1 and Table 2 the method was found to be precise and reproducible. Also the Interday variability has been performed and results were found reproducible. The measurement position observed is 4.64 (Table 3) which was an ideal position for the DLS measurement. Count rate observed was about 125 Kcps suggesting that sample is weak scattering. Attenuation used by the system is 11 representing the full laser power has been used for the determination. PDI value which is an indicator of the spread of particle sizes measured for the sample observed from 12 preparations ranges from 0.185 to 0.246 indicates the monodispersed nature of the sample. The Zavg. observed from 12 preparations was observed to be 1.76-2.40 nm with a mean value of 2.042, indicating that there is the absence of any particles above 5nm, hence the samples were observed to be free from any subvisible particulate matter which was likely to be present in the sample, a conclusion that is also supported by the count rate and attenuation data presenting the sample is weak scattering, hence free from particulate matter. I-MR (SPC) chart and Six pack capability matrix was applied to the Particle size and PDI value. To determine the special cause of variation or random behaviour all the eight Nelson’s rules have been applied into the Minitab. From the Figures nos. 7 and 8 the MR chart and I chart have been found stable and no special cause of variation was observed in the Z avg. and PDI suggesting the method is stable with all values lying in between ±3*standard deviation of mean Zavg, and PDI. Six pack capability analysis illustrated in Figures 9 and 10 demonstrate that the method is capable because Cpk>1.67, Ppk are nearly equal to Cpk indicating that the method is in statistical control.
Characterization of Solid Dispersions
Conclusion
The DLS method using the Malvern Zetasizer ultra has been developed to determine the particulate matter in a Biosimilar parenteral product (Calcitonin Salmon injection). From the statistical evaluation of the results the method is found to be stable and capable. The Z avg. and PDI values support the absence of any subvisible particulate matter in the sample which was further supported by the other quality attributes of DLS viz. count rate and attenuation. Considering the cost of biopharmaceutical products which is very high, relatively much less sample was used in this technique and since it is a non-invasive technique the same sample can be used for the other characterization tests. Hence the proposed method can be used to determine the particulate matter at submicron level in Biosimilar parenteral products.
Conflict of interest
None declared
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Authors
Akhilesh Kumar Kuril¹ K Saravanan²
¹Bhagwant University, Sikar Road, Ajmer, Rajasthan, India
453771, Madhya Pradesh, India
* Corresponding author:
¹*Akhilesh Kumar Kuril
1Bhagwant University
Sikar Road, Ajmer, Rajasthan, India
Email: gentleakhilesh@gmail.com
Co-author e-mail
K Saravanan
Email: kalyansar_mith@yahoo.co.in
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