In Quality Control (QC) procedures many pharmaceutical companies rely on microscope-based analyses to identify, qualify and or quantify solid particles meeting specified size criteria. A specification for QC analysis is agreed with the competent authority in the country where the product is to be marketed. For example the specification may limit the number of solid particles to 90% < 25 µm in length and 100% less than 100 µm in length. The method will also generally define the quantity of product to be used in the test. In practice, the sample is scanned by the analyst and an estimate of the total number of particles is made along with the percentage of solid particles meeting the specification. The samples must pass the analyses for the batch of product to be released for further processing or sale.
Often such manual microscope methods involve time-consuming procedures for the analyst and can be open to human subjectivity variations from analyst to analyst.
The Morphologi can automate such methods thus speeding up the analysis time, freeing-up the operators time and reducing error due to human subjectivity and/or fatigue. Additionally the system can distinguish particles according to their shape and classifications can be set up to identify the number or proportion of, for example, spherical and needle-shaped particles in a sample.
In Quality Control (QC) procedures many pharmaceutical companies rely on microscope-based analyses to identify, qualify and or quantify solid particles meeting specified size criteria. A specification for QC analysis is agreed with the competent authority in the country where the product is to be marketed. For example the specification may limit the number of solid particles to 90% < 25 µm in length and 100% less than 100 µm in length. The method will also generally define the quantity of product to be used in the test. In practice, the sample is scanned by the analyst and an estimate of the total number of particles is made along with the percentage of solid particles meeting the specification. The samples must pass the analyses for the batch of product to be released for further processing or sale.
Often such manual microscope methods involve time-consuming procedures for the analyst and can be open to human subjectivity variations from analyst to analyst.
The Morphologi can automate such methods thus speeding up the analysis time, freeing-up the operators time and reducing error due to human subjectivity and/or fatigue. Additionally the system can distinguish particles according to their shape and classifications can be set up to identify the number or proportion of, for example, spherical and needle-shaped particles in a sample.
A specific example where the Morphologi is being used to automate traditional manual microscopy quality control procedures involves monitoring the number of needle-shaped particles of an active pharmaceutical ingredient (API) in a pharmaceutical ointment. In this particular commercially available ointment one of the actives is known to form long, thin needle-shaped crystals in equilibrium with active dissolved in the ointment (Figure 1).
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Microscopy based quality control specifications for this product requires that in a 3 mg sample there must be no particles longer than 250 µm in length and no more than 100 particles can be longer than 100 µm. The number of particles between 50 and 100 µm long are also counted for information purposes. Current manual microscopy analysis can take well over two hours per sample and is subject to human bias. Automating the measurement on the Morphologi reduced sample analysis time to less than 15 minutes. The needle-shaped crystals are identified, measured, counted and classified in a manner free from human subjectivity. An additional advantage to the Morphologi is that a permanent record is kept of all particles analysed whereas in manual microscopy only a small but representative number of fields may be photographed.
The base of the ointment is a paraffin media which makes identification of the needle-shaped particles difficult by microscopy at room temperature. Heating the sample melts the paraffin base allowing the crystals to be identified (Figure 1).
Current procedures involve placing 3 mg of sample on a coverslip and placing this on microscope slide so the ointment is between the slide and the coverslip. The sample is then heated to 65 °C on a hot plate to melt the paraffin base. The sample spreads out and completely covers the area under the coverslip. The prepared sample is then transferred to a microscope for manual analysis of the needle-like particles. Manual analysis of the sample involves the operator identifying and measuring the length of all particles longer than 50 µm. The latter involves scanning up and down the slide a total of 20 times and ensuring that particles at each scan edge are not counted twice. During the procedure the sample cools down and the paraffin base solidifies preventing identification of the crystals. Therefore, the sample has to be reheated several times during the analysis and when it is reheated it is possible the particles will move slightly. Every time the slide is reheated, the analyst has to remember where they were on the slide so as to avoid missing a particle or counting some twice. The total time to measure a 3 mg sample is normally around 2 hours depending on the experience of the analyst and the number of crystals present in the sample.
The Morphologi particle characterization system is a high sensitivity automated image analysis instrument designed around a high specification microscope platform that measures and characterizes particles in terms of size and shape. It also has a classification feature that allows particles meeting specified criteria to be counted as being part of that class.
A Standard Operating Procedure (SOP) was set up to analyze the samples. An SOP contains all the software and hardware variables for the measurement so that every measurement is made in the same way. The use of an SOP eliminates variation from user to user, or site to site as it can easily be transferred between instruments.
The sample was prepared as previously described by heating it to 65 °C between a microscope slide and a coverslip on a hot plate. The slide was then placed on the Morphologi’s slide holder and a temperature controlled heat gun was used to maintain the sample temperature at 65 °C.
The instrument measures the samples according to the SOP by scanning the defined area whilst analyzing the particles in real time. The system also logs the x,y-coordinates of every particle ensuring that none are counted twice. The measurement time is less than 10 mins. The classification function was used to group the needle-like particles of different lengths together. The classes were defined as shown in Table 1.
| Class | Specification |
|---|---|
| ≥ 250 µm | Length ≥ 250 µm Aspect ratio < 0.33 |
| ≥ 100 < 250 µm | Length ≥ 100 < 250 µm Aspect ratio < 0.33 |
| ≥ 50 < 100 µm | Length ≥ 50 < 100 µm Aspect ratio < 0.33 |
Once the measurement is complete images of all particles are stored and the individual particles in each class can be viewed in the particle view.
If the operator wishes to look at any of the individual particles in more detail, for example at a higher magnification, they can click on the particle in the particle view and then return to it in the sample automatically from the logged x,y coordinates of every particle. If required a photograph of the field of view containing the particle of interest can be taken.
The results of all analyses are summarized in the records view and can be displayed or printed as a table in a customizable report (Table 2). Reports displaying the particles in specific classes are also customizable (Figure 2).
| Sample name | >250 µm | 100-250 µm | 50-100 µm |
|---|---|---|---|
| Sample 1 | 0 | 4 | 63 |
| Sample 2 | 0 | 3 | 126 |
| Sample 3 | 0 | 69 | 286 |
| Sample 4 | 0 | 4 | 26 |
| Sample 5 | 0 | 26 | 136 |
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The Morphologi particle characterization system provides the ability to automate Quality Control microscope methods that have previously been performed manually. The instrument can be set up to identify and count particles meeting certain size and/or shape criteria. This not only reduces measurement time but it also frees up the operator for other tasks whilst the analysis is being performed. Additionally it dramatically reduces variation in results due to human subjectivity.
