Product characterization is vital to the development of a robust lyophilized product and process for all formulation and process development. Lyophilized finished product should be evaluated for critical quality attributes (CQAs) specific to a lyophilized preparation. This can include physical appearance, quality of the constituted solution, residual moisture content and thermal properties.
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.
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.
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. An ATC probe may be used for automatic temperature compensation.
pH is recorded prior to lyophilization and during reconstitution of the finished product. Any shifts in pH warrant further investigation as they may be detrimental to the finished product.
Osmolarity is an important attribute depending upon the product route of administration. It 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.
One of the most vital analyses of lyophilized finished product involves moisture analysis by Karl Fisher Coulometric Titration. This analysis establishes the amount of moisture remaining in your product after lyophilization. The proper moisture range will be determined based on stability data. This analysis also demonstrates the success and efficiency of the freeze drying process and will determine the shelf life of your finished product.
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. Our state of the art instrument is routinely calibrated at temperatures that span the range of high temperature analysis.
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. This data is vital to determining if dried product will retain its state during storage, resulting in good long-term stability. HT DSC follows the current USP<891>.
Once thermal scans are 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.
Moisture analysis by LOD is accomplished by drying at elevated temperatures such as 60° or 105° C 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.