Donald Greiff

Protein structure and freeze-drying: The effects of residual moisture and gases☆

Abstract The drying of biologic materials by sublimation of ice in vacuo is not a single, simple process. The final product obtained is a function of the chemical and physical nature of the macromolecules present, the kinds and amounts of water molecules and their relation to the types and kinds of macromolecules, the influence of water on the conformation of the macromolecules, the gaseous environment in which dried preparations are placed and the physical principles regulating the process of freeze-drying.

Differential susceptibility of murine T and B lymphocytes to freeze-thaw and hypotonic shock

Abstract Experiments were carried out to determine if thymic-derived lymphocytes (T-cells) could be differentially damaged by hypotonic and/or freeze-thaw stress. The uptake of 3H-thymidine after stimulation of murine spleen cells with phytohemagglutinin-P (PHA-P) or bacterial endotoxin (LPS) was used as an indicator of recovery. Optimal freezing and thawing techniques showed that 100% of LPS-responsive cells could be recovered, compared to 65% of PHA-responsive cells. These differences could be increased by treatment of spleen cells with 0.17 m NH4Cl prior to freezing and thawing. This represented a recovery of 50% of LPS-responsive cells and less than 10% of PHA-responsive cells. A similar effect could be obtained by treating NH4Cl-treated spleen cells with distilled water prior to culture. It is hypothesized that T-cells are more susceptible to osmotic damage than B-cells due to their differences in membrane characteristics.

Thermal stability of freeze-dried mammalian interferons. Analysis of freeze-drying conditions and accelerated storage tests for murine interferon.

Conditions of Interferon processing were analyzed to select those that promote stability after freeze-drying. The effects of various preparative methods and treatment conditions were assessed by measuring the retention of biological activity by lyophilized interferon samples in two kinds of accelerated storage tests: the linear nonisothermal stability (LNS) test, a rapid method used for direct comparison of two or more preparations of interferon, and the multiple isothermal storage (MIS) test, a slow method requiring weeks to months to obtain data for the prediction of stability of a given preparation stored under various conditions. The most stable preparations of Newcastle-disease-virus-induced mouse L cell interferon were obtained using the following conditions: 1) perchlorate treatment to inactivate residual inducing virus, 2) nonspecific adsorption using zeolite for partial purification, 3) suspending medium of 0.5% bovine serum albumin in 0.1 m sodium phosphate buffer at pH 7, and 4) sublimation of ice in vacuo with a starting temperature of −30 °C to a final residual moisture of about 3%. The final product, reference reagent G002-904-511, was stable throughout the course of the LNS test. From an extensive MIS test, this reference interferon was predicted to lose 1000 units of activity in 110 years at 4 °C and 1000 units in 100 days at 37 °C. After 6 years of storage at 37 °C when the predicted residual activity would be about 20% of the original potency, 35% of initial interferon activity remained, confirming the usefulness of the short-term predictive test.

On the sealing of gas-filled glass ampoules.

Studies in our laboratories involved the placing of argon-filled hand-sealed glass ampoules (tip-, balloon-, or draw-sealed) containing biological materials dried by sublimation of ice in vacuo in water baths at elevated temperatures. Although these ampoules tested negative for “leakers,” many ampoules upon removal from the water bath contained beads of moisture. To determine if the water within the ampoules entered through openings in the closed ends, we used a laser imaging apparatus to examine the sealed ends. We carried out studies also on machine-sealed ampoules. Two modes of laser imaging were used: dark-field imaging and interference imaging. In tip-sealed ampoules, uniform, long channels were found in the gatherings of glass at the ends of the ampoules; the lengths of the channels were approximately ten times their diameters. In balloon-sealed ampoules, pore-like openings were found; the lengths of the pores were approximately four times their diameters. In draw-sealed ampoules, channels of small diameters were observed; the lengths of the open pathways were approximately 30 times their diameters. Based on the sizes of the images obtained with laser imaging, the magnifications used for photographic reproductions and the original measurements of the sealed ampoules we estimated the openings in tip-sealed and balloon-sealed ampoules to range from 5 to 8 μm and the channels in draw-sealed ampoules to be less than 3 μm in diameter, The diameters of the helix-like openings in machine-sealed ampoules were less than 5 μm. To prevent the migration of molecules into and out of ampoules, we sought for a barrier that could be interposed between the external environments of the ampoules. Many liquid formulations of natural and man-made elastomers were tested; neoprene dissolved in toluol was found best.

In vitro effects of cryoprotective agents on the response of murine T and B lymphoid subpopulations to mitogenic agents

Based on the ability of murine thymic derived lymphocytes (T-cells) to respond to phytohemagglutinin (PHA) and concanavalin-A (Con-A) and bursa equivalent or bone marrow derived lymphoid cells (B-cells) to respond to endotoxin (LPS) and pokeweed mitogen (PWM), a culture system was developed to study the effects of cryoprotective additives on the response of murine subpopulations to the various mitogens. It was found that glycerol and polyvinylpyrolidone (PVP) at various concentrations inhibited the response of spleen cells to all the mitogens. Dimethylsulfoxide (DMSO) at concentrations of 0.25–1.5% (0.03–0.19 m) significantly augmented the uptake of 3H-TdR in spleen cells stimulated with PHA-M or Con-A. Similar concentrations of DMSO depressed the response to LPS and PWM. Possible mechanisms are discussed.

Linear nonisothermal, single-step, stability studies of dried preparations of influenza virus

Abstract The rate constant of a reaction at elevated temperatures and the activation energies can be calculated from the values of the concentration of the reactant as a function of time. Measurements were made while the temperature of the reacting system was raised in accordance with the predetermined program. The method makes use of the Arrhenius equation, but it does not suffer from the disadvantage of the usual methods in which several experiments are done at fixed elevated temperatures.

Cryotolerance of selected sites on the surfaces of membranes of cells: II. A, B and H combining sites of human erythrocytes

Abstract Using a membrane adsorption test, based on the ability of the surfaces of red blood cells to react with group specific antibodies, the effects of rates of freezing, freezing and storage at low temperatures, and cyclic freezing and thawing on A, B, and H sites were measured. In a similar manner the ability of membranes to adsorb influenza virus was used to determine the cryotolerance of H sites. The washed red blood cells used in this study were suspended in phosphate buffer only or in phosphate buffer plus dimethylsulfoxide, glycerol, dextran, polyvinylpyrrolidone or hydroxyethyl starch. In general, the surfaces of membranes suspended in phosphate buffer only and exposed to the several freezing and storage procedures showed the greatest amount of damage. Following cyclic freezing (−76 °C) and thawing (+10 °C) the surfaces of erythrocyte membranes suspended in phosphate buffer plus dimethylsulfoxide or glycerol showed little damage to have occurred. Preparations suspended in phosphate buffer only or in polyvinylpyrrolidone showed the greatest amount of damage; increased damage occurred with each cycle of freezing and thawing. The damage to the surfaces of membranes suspended in dextran or hydroxyethyl starch were intermediate to the foregoing. Preparations of erythrocytes suspended in the several additives and cooled at 1, 15, or 100 °C/min showed either no damage or a small amount of damage to occur. In general, the least amount of damage occurred when preparations were cooled at 1 °C/min with additives present. All preparations were found to have small amounts of damage when stored at +4, −20, or −70 °C for 30 or 60 days. The least amount of change was found in preparations stored in 5% dimethylsulfoxide with progressively greater damage occurring in preparations stored in 10% glycerol, 3% dextran, 0.4% polyvinylpyrrolidone or 2% hydroxyethyl starch.

Functional activities of isolated lymphocytes following drying by sublimation of ice in vacuo: I. Rosette formation, stimulation by plant lectins (mitogens), and the mixed lymphocyte reaction

Abstract The following activities of isolated human lymphocytes were used for evaluating the effects of freezing and thawing and freeze-drying and rehydration on these cells: (a) spontaneous rosette formation, (b) responses to plant lectins (mitogens), and (c) the one-way mixed lymphocyte reaction. The successes achieved in drying of isolated lymphocytes by sublimation of ice in vacuo and rehydration with water with retention of the functions above, all of which appear to require living cells, were dependent upon a freeze-drying apparatus of unique design and the ability to freeze-dry suspending media containing dimethylsulfoxide. Best results were obtained when lymphocytes were: (a) isolated from blood collected in citrate-phosphate-dextrose (CPD); (b) suspended in Roswell Park Memorial Institute Medium-1640 (RPMI-1640) in sufficient amount to make 100%, 20% fetal calf serum, 8% serum albumin, 5% dimethylsulfoxide, and 1% L-glutamine; (c) cooled at approximately 1 °C/min from +4 to −25 °C and approximately 5 °C/min from −25 to −70 °C, and (d) rehydrated at low temperatures.

Freeze-Drying of Solutions of Serum Albumin Containing Dimethylsulfoxide,

Abstract : Although several publications have emphasized the inadvisability of drying biologic materials containing dimethylsulfoxide (DMSO) by sublimation of ice in vacuo, the current studies showed a relationship to exist between the relative concentrations of serum albumin, human, and dimethylsulfoxide for successful or unsuccessful freeze-drying of albumin-DMSO solutions. The cycle of freeze-drying for the successful drying of an albumin-DMSO solution was a modification of the cycle used for the successful drying of suspensions of measles virus by sublimation of ice in vacuo. Using nuclear magnetic resonance spectroscopy, a strong, sharp signal for DMSO was obtained in preparations of freeze-dried albumin-DMSO solutions rehydrated with deuterium oxide, D2O.

On the sealing of glass ampoules

Abstract : A method of sealing gas-filled glass ampules and the like to insure the closure of any microscopic channel existing between the outer and the inner surface thereof, and its product of manufacture is described. After the ampoule is filled and sealed, it and its contents are suitable for storing in ambient or low temperature, or for freezing, or freeze-drying. However, flame-sealing of the ampoules usually results in a small channel through the sealed tip. All typical flame-type sealing methods substantially close the ampoule but the microscopic channel still remains. The presence of the channel is undesirable since it permits gas exchange between the ampoule contents and the outside environment. Therefore, a polymeric coating of neoprene rubber dissolved in toluene or other suitable material is employed to seal the flame-sealed tips of the ampoules to assure closure of any existing channel. (Author)