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IVD Technology Magazine | IVDT Article Index

Originally published March, 1997

Effect of formulation on lyophilization, part 2

Thermal characteristics, the freeze dryer, secondary drying, and container selection

Thomas A. Jennings

To select constituents, consider their interactions during freezing and primary drying processes and possible reactions during secondary drying.

Note: This is the second part of a two-part article. Part one is also available for on-line viewing.

Thermal properties determine the suitability of a formulation for lyophilization. The main function of thermal analysis is to provide information to form a completely frozen matrix and the required product temperature and chamber pressure for conducting primary drying during lyophilization. Such information concerns the freezing characteristics of the water constituent and the degree of crystallization, collapse temperature, and any phase change in the interstitial region of the matrix.

Thermal Analysis of the Formulation

Method. Several analytical techniques determine the thermal characteristics of a formulation.1–7 This author prefers techniques known as D2 and differential thermal analysis (DTA) (protected by U.S. Patent No. 4,327,573). The analysis is conducted at the specified fill-volume in the containers that will be used to lyophilize and protect the formulation from the environment. With this method, we observe interactions between the container and the formulation.

The first of the two analytical techniques, D2, is defined as

It compares electrical resistivity of the reference process water (i) and formulation (s) during the freezing and thawing processes. The use of a water reference separates changes in the resistivity of the formulation (from the ice crystals) from those in the interstitial matrix region. This increases the sensitivity to detect the collapse temperature. The measurement of the resistance of the water reference (Ri) and of the resistance of the formulation (Rs) can be substituted for the resistivity terms if there is agreement with the cell constants of the resistance probes. We determine the collapse temperature, during the warming process, from the intersection of the line where D2 is equal to 1 or to a constant value (D2'), and the extrapolated value of D2 is from a least-squares regression analysis of the data points where D2 is greater than 1 or D2 is greater than D2'.

We derive phase changes in the formulation associated with the degree of supercooling and presence of interstitial region eutectic mixtures from the DTA. The results can be compared to simultaneous changes in the electrical resistance. DTA allows for the use of two reference materials: process water and methanol. From these references, we measure the degree of crystallization (D) in the formulation and the presence of any phase change that may occur in the interstitial region during freezing or warming.3,8

Data in graphic and tabular form aid interpretation. The water reference serves as not only a reference for the analysis but also a check on the accuracy of the temperature measurements near analysis completion.

Effect of Formulation on Thermal Properties

Because of the large variety of active ingredients and the countless possible combinations in composition and concentration of the excipients, we cannot select a typical formulation. For simplicity, we chose a 3% (w/v) aqueous formulation of lactose (D-[+]-lactose monohydrate powder baker analyzed ACS reagent [product #2248] supplied by Mallinckrodt Baker, Inc., Phillipsburg, NJ) and an isotonic 3% lactose solution (0.9% NaCl solution prepared from deionized water; NaCl certified ACS was supplied by Fisher Scientific Co., Pittsburgh).

The effect the composition had on the thermal properties of the above formulations was demonstrated by comparison of the thermal properties of the 3% lactose solution with those of an isotonic 3% lactose solution. Such a comparison of these relatively simple formulations illustrates the complex constituent interactions during freezing. The key thermal properties of these analyses are shown in Table I.


Table I. Comparison of key thermal properties of 3% (w/v) lactose solution and an isotonic 3% (w/v) lactose solution contained in a 5-ml clear glass tubing vial with 2-ml fill-volume.

The degree of supercooling came from an examination of the tabular data during the freezing process. The observed 8°C of supercooling for lactose and the isotonic lactose solution showed that it was sufficient, with the use of a proper cooling function, to produce a uniform matrix. We estimated the freezing of the matrix when D2 approached 1 or some constant value. The D2 for the lactose solution had a value of 1 at temperatures below –20°C, and the matrix was considered completely frozen. For the isotonic lactose solution, temperatures below –57°C were required before the value of D2 approached 1 and the frozen state was obtained.

Table I also shows that the addition of sodium chloride to the lactose solution resulted in a significant change in the collapse temperature from –19°C, agreeing with the collapse temperature for a lactose solution reported by others,9 to a value of –55°C. A second thermal analysis of the isotonic lactose solution indicated a collapse temperature of –52°C. Figures 1 and 2 graphically present the change caused by the addition of the isotonic solution.


Figure 1. Comparison plot of D2 as a function of the sample temperature, during the warming process, for a 3% lactose solution (red) and 3% lactose + saline solution (blue).


Figure 2. Comparison plot of the resistance (in kilohms) as a function of the sample temperature, during the warming process, for 3% lactose solution (red) and an isotonic 3% lactose solution (blue).

The increase in the D2 values with temperature indicates an increase in the mobility of the water in the interstitial region. Figure 2 further illustrates the effect of the addition of a saline solution to the 3% lactose solution. A decrease in the electrical resistance of the sample shows an increase in the mobility of the water in the interstitial region. The mobility of the water in the isotonic 3% lactose solution occurs at a significantly lower temperature than that of the 3% lactose solution. Hence, the addition of the NaCl to the lactose solution resulted in an interstitial region with significantly different thermal properties.

A glassy interstitial region typically has a collapse temperature that is not well defined, thus suggesting a frequency distribution of collapse temperatures for a given formulation. Such a frequency distribution could stem from the nonstoichiometric formation of the glassy states in the interstitial region. We observed additional evidence for this nonstoichiometric nature from the determination of the collapse temperature for 17 thermal analyses of a 3% (w/v) solution prepared from a source of anhydrous lactose (alpha-lactose, certified ACS, lot 746822, supplied by Fisher Scientific Co.). These results further support the hypothesis that during the freezing process involving the formation of glassy systems, some form of frequency distribution of the collapse temperatures may exist in the interstitial region of the matrix.


Figure 3. A comparison of the differential thermal analysis (DTA) thermograph for a 3% lactose solution (red) and an isotonic 3% lactose solution (blue). The grid spacing for the T (Ts–Tr) is 2°C. Ts refers to the sample temperature; Tr and reference temperature (°C) refer to the methanol reference temperature.

Figure 3 compares the DTA thermograph for the 3% lactose solution and the 3% lactose + saline solution during warming. There was no significant endothermic peak at temperatures below –10°C for the lactose solution nor for the isotonic lactose solution. The lack of an endothermic peak indicates that, during the formation of the frozen matrix, the concentration of the saline solution did not reach the required 23% NaCl concentration necessary for formation of the eutectic mixture. In the absence of a phase change in the interstitial region, the excipient in that region is in a glassy state.10

To measure the degree of crystallization, we took the ratio of the area under the endothermic peak for the melting of the sample, to the peak area for the melting of the water reference. The quantity of water in the water reference was adjusted to equal that in the sample. The degree of crystallization for the lactose solution (Table I) was 0.90, which indicates that 10% of the water in the formulation did not form ice crystals as a result of freezing. The addition of the NaCl to the lactose solution reduced the degree of crystallization to a value of 0.82. Thus, significantly less ice formed during freezing. The uncrystallized water is part of the glassy system in the interstitial region.

Effect of Thermal Properties on Freezing and Primary Drying

Freezing. The 3% lactose solution was completely frozen at temperatures below –30°C. However, to obtain a completely frozen matrix of an isotonic 3% lactose solution, a product temperature below –55°C is required. Thus, depending on the dimensions of the container and quantity of the fill-volume, obtaining a completely frozen matrix for the isotonic 3% lactose solution could require significantly more time to reach a frozen state.

Primary Drying. The rate of primary drying, related to the heat transfer rate into the container,11,12 is given by

Q = Co (Tr – Ts) cal sec–1

where Co is the heat transfer coefficient that is dependent on the composition and configuration of the container, Tr is the temperature of a reference container, and Ts is the temperature of the container containing the 3% lactose solution. The value of Co varies depending on the composition, configuration of the container, and chamber pressure. The estimated range of Co values is from 0.0001 cal°C–1sec–1 to 0.0500 cal°C–1sec–1. Here we assume that Co has a value of 0.005 cal°C–1sec–1 and that the heat of ice crystal sublimation is 700 cal/g.

For lyophilization in the 3% lactose solution, the product temperature in the frozen matrix must be significantly less than that of the collapse temperature. For sublimation, the chamber pressure must be less than that of the vapor pressure of ice (–25°C or 460 mtorr). By selecting a chamber pressure of 100 mtorr, sublimation occurs on the surface of the cake, and the initial ice temperature on the surface is about –40°C. By maintaining the pressure in the chamber at 100 mtorr with a gas bleed, the temperature of the frozen matrix is maintained at about –25°C by an increase in the shelf-surface temperature. We assume, for the purposes of illustration, that the shelf-surface temperature during primary drying is maintained at +10°C. Under these conditions, Tr has a value approaching +10°C; and Ts, –25°C. From the second equation provided above and the given value for Co, Q approaches 175 mcal sec–1 (mcal denotes millicalories). For a 5-ml fill-volume of a 3% (w/v) lactose solution, the approximate time to complete the primary drying is 5.2 hours.

The lyophilization of 5 ml of the isotonic 3% lactose solution requires the solution to be frozen to –60°C. The vapor pressure of ice at –60°C is about 8 mtorr. Therefore, the chamber pressure must be reduced to about 5 mtorr, at which the temperature of the ice-gas interface approaches –63°C (5 mtorr is assumed to be the ultimate lowest chamber pressure of the freeze dryer where the pumping speed of the vacuum pump approaches 0).

For a chamber pressure of 5 mtorr, the shelf-surface temperature necessary to maintain a product temperature of –60°C would be about –52°C. Based on the first equation provided above and a (Tr – Ts) value of 8°C, the primary drying for a 5-ml fill-volume of the isotonic 3% lactose solution requires 23.6 hours if the Co for the container still has a value of 0.005 cal°C–1sec–1. If the value of Co were reduced (a result of the lower chamber pressure of 5 mtorr) to 0.0025 cal°C–1sec–1, the time to complete the primary drying process would be extended to 46 hours.

The above example shows that the addition of a saline solution to a 3% lactose solution can not only increase the time required to complete the freezing but may also more than quadruple the time for completion of the isotonic lactose solution's primary drying.

Effects on Freeze Dryer Design, Construction, and Cost

The operating conditions of the freeze dryer for the 3% lactose and the isotonic 3% lactose solution are shown in Table II. The table indicates that because the freezing of a 3% lactose solution requires a temperature of only –30°C, the refrigeration system in most currently manufactured freeze dryers would be adequate. The isotonic 3% lactose solution requires additional refrigeration capacity to obtain shelf-surface temperatures of –65°C. Besides the cost for the additional refrigeration capacity, the manufacturer of the freeze dryer may be required to increase the horsepower of the circulating pump to meet required temperature variations across the shelf and from shelf to shelf.


Table II. Comparison of basic freeze-drying equipment operating conditions for the 3% lactose and isotonic 3% lactose solutions based on thermal properties listed in Table I.

An operating condenser temperature of –60°C is typical for most commercial freeze dryers, but the condenser temperature of –80°C, necessary for the isotonic solution, requires a more expensive refrigeration system.

For most dryers, the auxiliary refrigeration system has the capacity to maintain the shelf-surface temperature at –35°C and hence would have no difficulty maintaining the +10°C shelf-surface temperature for the 3% lactose solution. To maintain the required temperature of –52°C for the isotonic solution, a significantly larger auxiliary compressor is needed.

The 100-mtorr requirement for the primary drying of the 3% lactose solution is a pressure easily obtained and maintained by most commercial freeze dryers. The required chamber pressure of 5 mtorr for primary drying of the isotonic solution presents two problems: first, the additional pumping capacity required for the chamber to routinely achieve such pressure; and, second, the fact that at 100 mtorr the gas flow approaches viscous flow conditions and affects the diameter of the valve between the chamber and condenser. At a pressure of 5 mtorr, the gas flow becomes molecular, and the dimensions of the valve between the chamber and condenser must be greatly increased to achieve the necessary vapor throughput for the system.13

Conducting primary drying at 5 mtorr for the isotonic solution may require special design considerations for the condenser to prevent excessive ice build-up on the dryer's front surfaces. Back- streaming of hydrocarbons from the mechanical pumping system begins at about 100 mtorr, but becomes much greater at a pressure of 5 mtorr.14 We eliminate the backstreaming of hydrocarbon vapors for the primary drying of the isotonic solution by installing a hydrocarbon trap between the mechanical pump and the condenser chamber, or by using an oilless mechanical pumping system.15

Thus, additional refrigeration capacity for the shelves and the condenser, a larger-diameter main vacuum valve for the pumping system, and a special condenser system configuration make the freeze dryer for the isotonic 3% lactose solution far more costly than that for the lyophilization of the 3% lactose solution.

Effect of the Formulation on Secondary Drying

The secondary drying process reduces the free water in the product to levels no longer supporting biological growth or chemical reactions. The required range of free water necessary for stability will be dependent not only on the nature of the active ingredient but also on the selection of the excipients.

Consider that the active ingredient is a protein requiring an excipient to prevent covalent bonding between the protein molecules or to prevent overdrying. An excipient such as mannitol may be inadequate; it tends to form crystals during freezing16 and to leave the protein exposed on the mannitol. Sucrose, however, tends to form glassy systems10 during freezing and surrounds the protein molecules. In addition, lyophilized sucrose is amorphous and hygroscopic. As a result of incomplete secondary drying or high moisture content in the closure, dissolution of the cake may occur. If stability of the active ingredient requires moisture levels of less than 1%, mannitol would be an even better excipient.

A low solid content in a formulation can produce poor supporting structure with cake breakup during drying. A formulation with a relatively high solid content of more than 20% may form such a dense cake structure that transport of water vapor from the cake during secondary drying is impeded.17 For dense cake structure, the moisture level can be reduced by increasing product temperature, decreasing chamber pressure, or doing both. Prolonged exposure to low chamber pressures (<50 mtorr), either at elevated or at ambient temperatures, exposes the final product to possible contamination from the hydrocarbon backstreaming of the mechanical pump.

The moisture of dense cakes has been successfully reduced by a series of gas purges. Although varying from product to product, moisture values of less than 1% are possible after about 10 purges. Use caution with a gas purge during secondary drying on formulations that have poor self-supporting structures or if the solid content of the formulation is less than 2%.

Until recently, interactions of the active ingredient or of the excipients were thought to occur either during freezing or primary drying. Calorimetric monitoring of lyophilization using the drying process monitor (protected by U.S. Patent No. 5,280,678) during secondary drying showed that exothermic reactions between constituents are responsible for producing turbid solutions on reconstitution.12 Therefore, selection of constituents for a formulation must also consider their compatibility during secondary drying.

Effect of Formulation on Container Selection

The most pronounced aspects of the formulation (sensitivity of the active ingredient to light and solubility) determine the selection of the container. Effects of radiation can be reduced by amber vials. However, low solubility of an active ingredient in water presents special problems.

Active ingredients with poor solubility in water need an excessive fill-volume and a bulking excipient to provide adequate cake structure. One way to reduce required fill-volume is increasing solubility of the active ingredient by the addition of tertiary butyl alcohol (TBA). TBA has freezing and vapor properties similar to those of water and, for the most part, will not affect the collapse temperature as do other alcohols such as methanol or ethanol.

Conclusion

The selection of formulation constituents can no longer be limited to the formation of a stable aqueous environment for the active ingredient. Consideration must be given to the impact various constituents have on the thermal properties of the formulation. Low collapse temperature or degree of crystallization affects not only manufacturing productivity, but also design, construction, and cost of freeze-drying equipment. Formation of glassy states in the matrix interstitial region may be nonstoichiometric and result in a frequency distribution of collapse temperatures.

References

1. MacKenzie AP, "Collapse during Freeze-Drying--Qualitative and Quantitative Aspects," in Freeze Drying and Advanced Food Technology, Goldblith SA, Rey L, and Rothmayr WW (eds), New York, Academic Press, pp 277–306, 1975.

2. Jennings TA, "The Effect of Resistivity Probe Design on the Measurement of the Freezing Temperature of Lyophilized Formulations," J Parent Drug Assoc, 34:109–126, 1980.

3. Jennings TA, "Thermal-Analysis Instrumentation for Lyophilization," Med Dev Diag Indust, 2(11):48–57, 1980.

4. Williams NA, and Poli GP, "The Lyophilization of Pharmaceuticals: A Literature Review," J Parent Sci Tech, 38:48, 1984.

5. Sheu S, and Willson J, "The Generalized Form of the Electrical Resistance for a Frozen Matrix of an Aqueous Solution," J Parent Sci Tech, 47:180, 1993.

6. Gatlin L, and DeLuca PP, "A Study of the Phase Transitions in Frozen Solutions by Differential Scanning Calorimetry," Parent Drug Assoc, 34:398, 1980.

7. Rey L, "Fundamental Aspects of Lyophilization," in Aspects Théoriques et Industriels de la Lyophilisation, Rey L (ed), Paris, Herman, pp 23–43, 1964.

8. Jennings TA, "Lyophilization Seminar" (Notes), Conshohocken, PA, Phase Technologies, p 197, 1994.

9. Karel M, "Heat and Mass Transfer in Freeze-Drying," in Freeze Drying and Advanced Food Technology, Goldblith SA, Rey L, and Rothmayr WW (eds), New York, Academic Press, pp 177–202, 1975.

10. Greaves RIN, "Fundamental Aspects of Freeze-Drying Bacterial and Living Cells," in Aspects Théoriques et Industriels de la Lyophilisation, Rey L (ed), Paris, Herman, pp 407–410, 1964.

11. Jennings TA, "Discussion of Primary Drying during Lyophilization," J Parent Sci Tech, 42:118–121, 1988.

12. Jennings TA, and Duan H, "Calorimetric Monitoring of Lyophilization," PDA J Pharm Sci Tech, 49:272–282, 1995.

13. Dushman S, Scientific Foundations of Vacuum Technology (2nd ed), Lafferty JM (ed), New York, Wiley, pp 80–111, 1962.

14. Amoignon J, "Physico-Chemical Contamination of Freeze Dried Pharmaceutical and Biological Products," in Freeze Drying and Advanced Food Technology, Goldblith SA, Rey L, and Rothmayr WW (eds), New York, Academic Press, pp 445–460, 1975.

15. Wycliffe H, "Mechanical High-Vacuum Pumps with an Oil-Free Swept Volume," J Vac Sci Tech, 5A:2608–2611, 1987.

16. Williams NA, Lee Y, Polli GP, et al., "The Effects of Cooling Rate on Solid Phase Transitions and Associated Vial Breakage Occurring in Frozen Mannitol Solutions," J Parent Sci Tech, 40:135–141, 1986.

17. Pikal MJ, Roy ML, and Shah S, "Mass and Heat Transfer in Vial Freeze-Drying of Pharmaceuticals: Role of the Vial," J Pharm Sci, 73:1224–1237, 1984.

Thomas A. Jennings, PhD, is CEO, Phase Technologies, Inc. (Conshohocken, PA).

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