Protection of a model protein, lactate dehydrogenase by encapsulation in liposome.

The objective of this study was to determine if encapsulating a model protein, lactate dehydrogenase (LDH), inside liposomes can protect it during storage, upon freeze-thaw cycles and during freeze-drying. LDH, which undergoes cold denaturation, was entrapped insides liposomes prepared using modification of Bangham's method. It was found that when LDH was entrapped in liposomes and subjected to a single freeze-thaw treatment, there was about 7 times more recovery of activity than when LDH was free in solution. Similarly, LDH encapsulated in liposomes showed 60% recovery after freeze-drying compared to 1-2% recovery for the unentrapped LDH. The role of phospholipid content, residual moisture content and protein content were evaluated. Possible mechanism(s) of cold temperature inactivation of proteins and role of liposomes in preventing such cryodenaturation will be discussed in light of these results.

Ability of laboratory methods to predict in-use efficacy of antimicrobial preservatives in an experimental cosmetic.

Volume 60, no. 12, p. 4553: a present address for S. J. Wells should be given, as follows: (dag) Present address: Cadbury Beverages North America, Trumbull, CT 06611. [This corrects the article on p. 4553 in vol. 60.].

Ability of laboratory methods to predict in-use efficacy of antimicrobial preservatives in an experimental cosmetic.

The abilities of nine antimicrobial systems to preserve an experimental water-based cosmetic formulation were evaluated by six microbiological challenge tests: the U.S. Pharmacopeia test; the British Pharmacopeia test; the Cosmetic, Toiletry, and Fragrance Association test; the rapid screen test; the sequential challenge test; and the post-use test. The antimicrobial systems contained various combinations and amounts of two parabens and a quaternary compound in order to provide a broad range of preservation. The results obtained were compared with the abilities of the formulations to support maintenance and growth of microorganisms in microfloras obtained from human axilla areas and finger skin during an 8-week simulated in-use test. Without statistical analysis all of the tests predicted the results obtained with well-preserved or poorly preserved formulations. The rapid screen test was the best test for predicting differences at intermediate levels of preservation. Statistically, all of the tests were equivalent predictors of preservation efficacy in the in-use test (P = 0.05). At the P = 0.10 level, only the U.S. Pharmacopeia, British Pharmacopeia, rapid screen, Cosmetic, Toiletry, and Fragrance Association tests were significantly predictive. The results of prediction by a test, based on the preservative levels used, agreed well with the in-use test results (P = 0.01). A total of 20% of the formulations that contained excessive microbial levels contained human axilla microorganisms. The levels of preservation in failed products were similar to the levels of preservation in unused controls.

Protection of lactate dehydrogenase against protease digestion by encapsulation in liposomes.

Lactate dehydrogenase (LDH) was entrapped in phosphatidylcholine liposomes to evaluate the protective effect of liposomes against protease digestion. Three different preparations of LDH either encapsulated in liposomes, unencapsulated in liposomes or in the absence of liposomes were incubated with the protease trypsin. The loss of LDH activity was measured at intervals over a 12-hour period. The degradation rate of LDH was found to be the same when LDH was unencapsulated in the presence or absence of liposomes. However, when LDH was entrapped in liposomes the degradation rate was 4 to 24 folds slower. This finding indicates that encapsulation of a protein in a liposome protects the protein from the degrading effects of a protease enzyme.

Membrane filtration of pharmaceutical solutions.

The principles and uses of membrane filtration in the preparation of pharmaceutical solutions are discussed. The preparation of pharmaceutical solutions often requires their passage through a thin polymeric membrane containing many tiny pores. The purpose is to remove viable and nonviable particles in order to clarify or sterilize the solution. Particles may be retained by sieving, entrapment, or electrostatic attraction. The largest pore size that will yield a sterile filtrate is 0.2 microns. Membrane filters are either hydrophobic or hydrophilic. The rate of flow through a filter is affected by the resistance of the filter, the viscosity of the solution, and pressure. Filters are commonly composed of mixed esters of cellulose, polysulfone, polyvinylidene difluoride, nylon 66, polycarbonate, or polytetrafluoroethylene. In selecting a membrane filter, a pharmacist must consider pore size, compatibility, fluid volume, particulate load, and the filter holder. Various tests are available to evaluate the integrity of filters. Filters are useful in testing end products for sterility. The use of an in-line filter during the administration of large-volume injectable solutions can prevent the introduction of particles, air, and microorganisms into the patient. Therapies in which a 0.2-micron filter may be contraindicated include lipid emulsions, low-dose infusions, low-volume infusions, drugs for which the pharmacologic properties are altered by the membrane filter, and drugs that adhere to the membrane. Pharmacists need an in-depth understanding of the principles of filtration, the characteristics of filters, and their use in the filtration of pharmaceutical solutions.

Freeze-thaw studies of a model protein, lactate dehydrogenase, in the presence of cryoprotectants.

The objective of this study was to investigate the behavior of lactate dehydrogenase (LDH) upon freezing and thawing, alone or in the presence of several selected cryoprotectants. Also, the influence of the freezing rate on retainment of LDH activity was investigated. It was observed that fast freezing caused less loss of LDH activity than slow freezing. The probable mechanisms of loss of activity after freeze-thaw cycles were discussed. Selected cryoprotectants were evaluated for their ability to protect LDH during freeze-thaw cycles. Surface tension and pH change measurements upon freezing of the cryoprotectant solutions were carried out. Based on the results of these experiments, a potential mechanism of cryoprotection has been developed.

Loss of LDH activity during membrane filtration.

Membrane filters rendered hydrophilic and composed of biphenyl polycarbonate, polyvinylidene fluoride (PVDF), acrylic copolymer, polysulfone, and mixed esters of cellulose were evaluated to determine which type of filter best can be used for the filtration of lactate dehydrogenase (LDH) solution. Also, the effect of the membrane pore size was evaluated. LDH solution was passed through the filters at a controlled flow rate, after which the filtrate was assayed for LDH activity and protein content using the Bradford method. Polycarbonate and PVDF filters generally showed low loss of protein, except 5 microns PVDF filters. Mixed esters of cellulose, acrylic copolymer, and polysulfone caused considerable loss of protein during passage of the LDH solution through the filter. Interestingly, it was also found that, generally, as the pore size increased the amount of protein loss decreased. However, 5 microns PVDF and mixed esters of cellulose filters showed more loss than their corresponding 0.65 microns and 3 microns pore size filters, respectively. In all cases, more protein was recovered in the filtrate fractions as the volume of LDH solution filtered was increased, suggesting that the mechanism of loss is adsorption and that the magnitude of loss is related to saturation of the matrix polymer surfaces.

Evaluation of closure integrity after multiple penetrations.

The purpose of this investigation was to simulate in-use testing of closures to determine how many doses could be reasonably withdrawn from a multiple dose vial of a product without compromising the integrity of the closure. Four types of studies were done: 1) a simulated in-use test, 2) a coring test development study, 3) effect of sterilization on coring, and 4) exploratory studies of closure leakage after multiple penetrations. A modified drill press was used for the studies. West 1888 and West 1535 closures were evaluated by the simulated in-use test using a 26G Needle. The results indicated that few particles were generated after 20 penetrations of both closures. There was a marked increase in particles after 30 penetrations. When sterilized closures were tested, it was found that West 1888 generated no particles after 10 insertions with 21G and 18G needles, but there was an increase in the number of particles after 20 insertions. With West 1535, particles were generated after only 10 insertions. Interestingly, autoclaved closures released fewer particles than closures that were not autoclaved. Leakage was observed only from West 850 closures and only under relatively high pressure differential conditions.

Dry heat inactivation of endotoxin on the surface of glass.

The thermal inactivation of three endotoxin preparations on the inner surface of glass capillary tubes was studied. The samples were exposed to precisely controlled dry heat conditions at study temperatures ranging from 170 degrees to 350 degrees C, and were assayed using the gel-clot method of the Limulus Amebocyte Lysate test. Plots of the log of the amount of pyrogenic material remaining versus heating time revealed apparently biphasic destruction curves. The initial slopes were linear to a minimum 3-log unit reduction, and were followed by slower destruction rates for the terminal slopes. D values were calculated from the initial slopes of the destruction data, and Z values were estimated from the D values. The D and Z values were found to vary with the initial charged amounts of endotoxin. A second-order equation was found to be an inappropriate model for the inactivation process at temperatures between 170 degrees and 250 degrees C, but was found to be suitable for temperatures between 250 degrees and 325 degrees C. The data were successfully fit to a biexponential equation for all the temperatures studied. The overall inactivation rate of the endotoxin material formulated with fillers was apparently faster than that for the pure endotoxin preparations.

Recovery of endotoxin preparations from the surface of glass capillary tubes.

The gel-clot method of the Limulus Amebocyte Lysate (LAL) test was used to determine the recovery efficiency of four different endotoxin preparations from Type I glass capillary tubes. Each capillary tube was charged with 10,000 endotoxin units (EU) and vacuum-dried at 50 degrees C. Average recovery for the Whittaker M.A. Bioproducts Control Standard Endotoxin (CSE), which contained fillers, was 90% (80-100%), for the Difco Laboratories and Associates of Cape Cod endotoxins 47% (40-80% and 20-100%, respectively), and for the rough lipopolysaccharide 70% (40-100%). The presence of fillers appears to increase the percentage of endotoxin recovered and decrease the inconsistency in recovery results. The overall recovery rates for the pure endotoxin formulations were higher and slightly more consistent than those reported in previous studies.

Special considerations in the use of vertical laminar-flow workbenches.

The design, operation, and proper use of vertical laminar-flow workbenches are reviewed. Vertical-flow hoods are different from horizontal-flow units in several important ways that must be considered by operators who may have been trained to use the horizontal-flow type. Air in vertical-flow units provides practically no resistance to ingress of air propelled by body motions of the operator or passers-by or from nearby ventilation ducts. The HEPA-filtered air hits the work surface perpendicularly and must travel horizontally to reach the exhaust ducts; thus, manipulations should not be performed close to the work surface. Turbulence patterns around objects in the vertical flow hood will be different from that in horizontal-flow units. Manipulative technique is also different in a vertical-flow hood. Supplies may be arranged to the sides and the back of the area in which manipulations will be performed. Items not sterile, including fingers and hands, must be kept downstream from critical sites. Masks do not have to be worn because of the hood's glass panel. Operators must be cognizant of basic differences between vertical- and horizontal-flow hoods.

Qaulity-control plan for intravenous admixture programs. II: Validation of operator technique.

A plan for the validation of aseptic-operator technique in i.v. admixture programs and two test methods for evaluating the plan are proposed. After a new operator has been trained, the plan involves qualification of the operator through the preparation of statistically valid samples, to be followed by the random selection of samples for in-process monitoring. To test the plan, trypticase soy broth transfers were used in one hospital and Addi-Chek (Millipore Corp.) filtrations were used in another. The participants, all trained operators, initially prepared 40 test samples as a validation step. The finding of no microbial growth in these test samples permitted continuation into the monitoring phase, during which test samples were prepared randomly, one test sample out of every 25 i.v. admixtures prepared for patient use. All samples were negative for microbial growth, indicating that the operators maintained aseptic technique. These findings give evidence that the proposed testing plan is valid. The authors propose the plan as a phase of a quality control program, based on valid statistical principles, to give assurance that i.v. room operators are qualified to prepare sterile parenteral medications.

Quality-control plan for intravenous admixture programs. I: Visual inspection of solutions and environmental testing.

Two components of a model quality-control plan for intravenous admixture services--a 100% visual inspection and an environmental testing program--were developed and evaluated in two hospitals. Visual inspections are recorded by technicians on two forms that detail the types of solution, container, and device defects that should be checked. One form covers stock solutions and supplies, and the other covers prepared solutions. The environmental testing program uses two forms to record performance and results of routine microbial monitoring. The detailed quality-control forms are presented. Procedures for handling defective items and contamination problems are discussed. Evaluation of the visual and environmental plans showed they provided a systematic, workable, and modest-cost method of assuring and documenting conditions in an i.v. admixture service. To assure competent preparation, use and distribution of high-quality i.v. solutions, both components of the quality-control system must be performed on a routine basis.