Physical inspection evaluates uniformity of appearance in color, texture, shape, structure and provides insight into the relative effects of processing. Using an inspection light box with white and black backgrounds, each sample is viewed at the bottom, sides and top surface of the cake while rotating the container to view all sides. The extent, range and/or consistency for each of the aforementioned attributes are considered and recorded. Color can be characterized as intensity of the color, hue or tint indicating the color tone and shade that reflects lightness to darkness of the color. Product structure can be indicative on more of a microscopic scale described as dense or light, granular or geometric, crystalline looking shapes or composition that make up the arrangement, configuration, pattern, or organization. Texture can range from smooth or fine, appearing like powdered sugar or chalk with indistinguishable finite structure, to a course texture where structure is easily observed. Course texture has a makeup that appears like a collection of open, porous material as in a composition of particles, or assembly of crystalline or granular appearing material.
Reconstitution of Solution
Attributes of the constituted solution are described in the current USP <1>, Injections. The requirements of a constituted solution state that there is no visible insoluble material and the solution is no less clear than the diluent after a pre-determined amount of time. The typical volume for reconstitution returns the product to the same volume and concentration as the starting product used for filling the bulk solution. However, some products are reconstituted at a different concentration and volume than that used for the filled solution. Products may be reconstituted with diluents other than sterile Water for Injection. They may include sterile Normal Saline (0.9% NaCl) and Bacteriostatic Water for Injection and Bacteriostatic Normal Saline. The diluent used may have an influence on the reconstitution time or attributes of the constituted solution.
Once the appropriate amount of diluent is determined, the required volume of solution for reconstitution is drawn up into a syringe. The diluent is then extruded into the center of the dried cake and the timer is started. The product is inspected at approximately five second intervals in a light box to verify the absence of any insoluble material and clarity of the solution. The solution, once fully reconstituted, is characterized as being clear, colorless, hazy, opaque and/or cloudy. Particles, if present, are classified in a range from fine insolubles to coarse fibers.
Both freshly formulated solution and reconstituted solution is pH measured per USP<791>, pH. Standardization with two or three pH buffers that bracket the expected sample range are performed prior to use. The pH buffers chosen are no greater than three pH units, but no less than two pH units apart (e.g. 4.01, 7.00, and 10.01). An ATC probe may be used for automatic temperature compensation. If an ATC probe is not available and the sample to be measured is colder or hotter than room temperature, the buffers are chilled or heated accordingly.
A quantity of the sample solution sufficient enough to cover the pH probe sensor and any reference junction on the side of the probe is dispensed into a suitable container. The solution is gently swirled and then allowed to stand still and stabilize to a constant value over a period of at least 15 seconds, at which point, the displayed pH value is recorded.
Osmolarity is a measurement of the total solute concentration in a liquid. It is written as moles of solute present/Kg of solvent (or as millimoles of solute present per liter of solution) to measure the freezing point of the sample solution. The instrument utilizes a high-precision, electronic thermometer to sense the sample temperature, to control the degree of supercooling and freeze induction, and to measure the freezing point of the sample.
A standard check is performed before each use with a sample of Clinitrol 290 Reference Solution. The sampler plunger is depressed and the tip inserted to load a 20µl sample. The sampler barrel is loaded into the osmometer, and the entire operating cradle is pushed down into the unit. The instrument eventually reports the osmolarity that can then be recorded.
Coulometric Karl Fischer Titration
Moisture testing follows widely accepted, conventional methods outlined under USP<921>, Water Determination. A general standard method is described in the section below. Alternate methods are also developed on an as-needed basis.
The initial dried sample and container are weighed. A solvent extraction method with anhydrous methanol, special reagent, A.C.S. injected into the container is used to suspend and dissolve the dried substance. The samples are then allowed to soak for a predefined time in order to extract the moisture in the product. An aliquot is removed; the volume is measured and injected into the reaction vessel of the KF instrument. Upon reaching an end point of the titration, the results are reported. The KF instrument resolves water content to micrograms. The empty container is weighed and the percentage moisture is calculated for the initial container contents.
Thermogravimetric Analysis (TGA) may be used as a corroborative method for residual moisture determination where the change in weight is attributed to the evolution of volatile substances, such as water. In addition, it can be used to determine physico-chemical changes as the specimen begins to decompose at elevated temperatures.
TGA monitors the change in weight of a material as a function of temperature or time. The analysis is performed following USP<891>, Thermal Analysis. Approximately 5 to 15 mg of solid material are placed in an open aluminum sample pan. The sample is analyzed using a warming rate of 10°C per minute to measure the weight loss across the temperature range. Nitrogen, NF is used to purge the sample continuously at a flow rate of 60 mL/minute. The instrument is routinely calibrated at temperatures that span the range of high temperature analysis.
During warming, weight loss up to a plateau at approximately 100°C is associated with the evolution of water in the sample. The onset of degradation is determined after the plateau at the temperature where significant weight loss begins to occur. The scan data is recorded and graphed simultaneously.
High Temperature Differential Scanning Calorimetry
High temperature differential scanning calorimetry (HT DSC) is used as a means of determining the thermal properties of solid materials. It provides useful information for evaluating the formulation and assessing behavior in the dried state. HT DSC follows the current USP<891>.
Approximately 5 to 15 mg of solution is placed in an open, aluminum sample pan. Two warming rates are utilized for each vial tested. Initially, a 10°C per minute rate is used to increase the sensitivity of the analysis. A second analysis is performed at a slower warming rate of 2°C per minute to improve the accuracy of the measured temperature. Nitrogen, NF, is used to purge the sample continuously at a flow rate of 50 mL/minute.
The lyophilized material is warmed to evaluate the thermal behavior at temperatures above ambient conditions. During warming, evolution or uptake of heat from the sample reflects the differences in energy as the sample undergoes a thermal event. The scan data is recorded and graphed simultaneously using the Advantage software. Once the scan is complete, calculations identify the temperature at the onset and peak of the observed thermal event. Temperatures, changes in enthalpy, and/or changes in specific heat are determined for the observed thermal events such as glass transitions (Tg’), crystallization and melting points.
Loss on Drying
Moisture analysis by LOD is accomplished by drying at elevated temperatures such as 60 or 105C under atmospheric or vacuum conditions for a specified time. Samples are weighed on a calibrated analytical balance before and after drying. A dedicated calibrated oven capable of operating at reduced pressure or using a nitrogen atmosphere is used for the drying. Testing can be done using covered glass vessels or directly in the finished product container. Results are calculated based upon the weight loss as compared to the initial contents and container.