Sucrose Formulations Research Articles

Effect of sorbitol and residual moisture on the stability of lyophilized antibodies: Implications for the mechanism of protein stabilization in the solid state

PurposeTo investigate the effect of plasticizers on the stability of protein formulations in the solid state and to apply these results to a study of mechanisms of protein stabilization by sugars in the solid sate. MethodsThe IgG1 antibody was formulated with either sucrose or trehalose alone or a mixture of sorbitol with sucrose or trehalose. After lyophilization, the pure protein and sucrose formulations were equilibrated at different relative humidities giving residual moistures from less than 1% to 5% for sucrose systems and up to 17% for pure protein systems. All the samples were stored at 50°C for up to1month and at 40 and 25°C for up to 6months. Aggregation and chemical degradation were monitored by size exclusion chromatography (SEC) and ion exchange chromatography (IEX), respectively. The secondary structure was characterized by FTIR using second derivative analysis of Amide I region. Structural relaxation times, τ, an indication of molecular mobility in the glassy matrix, were characterized using the thermal activity monitor (TAM). The τ values of the recombinant human monoclonal antibody (rhuMab) formulation with various water contents were also measured in this study and compared with stability data taken from the literature (Breen ED, Curley JG, Overcashier DE, Hsu CC, Shire SJ, 2001, Pharm Res 18:1345–1353). ResultsThe structural relaxation time, τ, decreased sharply with increasing water content. However, the stability data suggest a minimum in degradation rate at 2%–3% water content. Addition of a small amount of sorbitol to a sucrose-based formulation resulted in greater retention of native structure, smaller relaxation time, but improved stability. However, with the trehalose-based formulations, addition of sorbitol had no effect on protein structure (FTIR), but the decrease in relaxation time and the improvement in stability were qualitatively similar to the corresponding data obtained with the sucrose-based formulation. ConclusionGlass dynamics as measured by τ could not explain the stability results. Stability correlated best with the preservation of native structure for sucrose-based formulations, but with the trehalose-based formulation, neither structural relaxation time nor extent of native structure was predictive of stability. However, it is possible that the β-relaxations rather than the α-relaxation (i.e., the τ we measured) is critical to the stability. Plasticizers like glycerol may decrease τ for “α-motion” but increase τ for “β-motion” and stabilize proteins (Cicerone MT, Tellington A, Trost L, Sokolov A, 2003, BioProcess Inter 1:1–9). © 2005 Wiley-Liss, Inc. and the American Pharmacists Association

Measurement of the Kinetics of Protein Unfolding in Viscous Systems and Implications for Protein Stability in Freeze-Drying

The aim of the study is to determine the degree of coupling between protein unfolding rate and system viscosity at low temperatures in systems relevant to freeze-drying. The cold denaturation of both phosphoglycerate kinase (PGK) and beta-lactoglobulin were chosen as models for the protein unfolding kinetics study. The system viscosity was enhanced by adding stabilizers (such as sucrose), and denaturant (guanidine hydrochloride or urea) was added to balance the stabilizing effect of sucrose to maintain the cold denaturation temperature roughly constant. The protein unfolding kinetics were studied by both temperature-controlled tryptophan emission fluorescence spectroscopy and isothermal high-sensitivity modulated differential scanning calorimetry (MDSC) (Tzero). Viscometers were used to determine the system viscosity. To verify the predictions of structure based on protein unfolding dynamics, protein formulations were freeze-dried above the glass transition temperatures, and the protein structures in dry products were determined by fluorescence spectroscopy of reconstituted solids by extrapolation of the solution data to the time of reconstitution. Empirical equations describing the effect of sucrose and denaturant (urea and guanidine hydrochloride) on protein cold denaturation were developed based on DSC observations [X. C. Tang and M. J. Pikal. The Effects of Stabilizers and Denaturants on the Cold Denaturation Temperature of Proteins and Implications for Freeze-Drying. Pharm. Res. Submitted (2004)]. It was found that protein cold denaturation temperature can be maintained constant in system of increasing sucrose concentration by simultaneous addition of denaturants (urea and guanidine hydrochloride) using the empirical equations as a guide. System viscosities were found to increase dramatically with increasing sucrose concentration and decreasing temperature. The rate constants of protein unfolding (or the half-life of unfolding) below the cold denaturation temperature were determined by fitting the time dependence of either fluorescence spectroscopy peak position shift or DSC heat capacity increase to a first-order reversible kinetic model. The half-life of unfolding did slow considerably as system viscosity increased. The half-life of PGK unfolding, which was only 3.5 min in dilute buffer solution at -10 degrees C, was found to be about 200 min in 37% sucrose at the same temperature. Kinetics of protein unfolding are identical as measured by tryptophan fluorescence emission spectroscopy and by high-sensitivity modulated DSC. The coupling between protein unfolding kinetics and system viscosity for both proteins was significant with a stronger coupling with PGK than with beta-lactoglobulin. The half-lives of PGK and beta-lactoglobulin unfolding are estimated to be 5.5 x 10(11) and 2.2 years, respectively, even when they are freeze-dried in sucrose formulations 20 degrees C above Tg'. Thus, freeze-drying below Tg' should not be necessary to preserve the native conformation. In support of this conclusion, native PGK was obtained after the freeze-drying of PGK at a temperature more than 60 degrees C above the system Tg' in a thermodynamically unstable system during freeze-drying. Protein unfolding kinetics is highly coupled with system viscosity in high viscosity systems, and the coupling coefficients are protein dependent. Protein unfolding is very slow on the time scale of freeze-drying, even when the system is freeze-dried well above Tg'. Thus, it is not always necessary to freeze-dry protein formulations at temperature below Tg' to avoid protein unfolding. That is, protein formulations could be freeze-dried at product temperature far above the Tg', thereby allowing much shorter freeze-drying cycle times, with dry cake structure being maintained by the simultaneous use of a bulking agent and a disaccharide stabilizer.

The Nonsteady State Modeling of Freeze Drying: In-Process Product Temperature and Moisture Content Mapping and Pharmaceutical Product Quality Applications

Introduction. Theoretical models of the freeze-drying process are potentially useful to guide the design of a freeze-drying process as well as to obtain information not readily accessible by direct experimentation, such as moisture distribution and glass transition temperature, Tg, within a vial during processing. Previous models were either restricted to the steady state and/or to one-dimensional problems. While such models are useful, the restrictions seriously limit applications of the theory. An earlier work from these laboratories presented a nonsteady state, two-dimensional model (which becomes a three-dimensional model with an axis of symmetry) of sublimation and desorption that is quite versatile and allows the user to investigate a wide variety of heat and mass transfer problems in both primary and secondary drying. The earlier treatment focused on the mathematical details of the finite element formulation of the problem and on validation of the calculations. The objective of the current study is to provide the physical rational for the choice of boundary conditions, to validate the model by comparison of calculated results with experimental data, and to discuss several representative pharmaceutical applications. To validate the model and evaluate its utility in studying distribution of moisture and glass transition temperature in a representative product, calculations for a sucrose-based formulation were performed, and selected results were compared with experimental data. Theoretical Model. The model is based on a set of coupled differential equations resulting from constraints imposed by conservation of energy and mass, where numerical results are obtained using finite element analysis. Use of the model proceeds via a “modular software package” supported by Technalysis Inc. (Passage™/Freeze Drying). This package allows the user to define the problem by inputing shelf temperature, chamber pressure, container properties, product properties, and numerical analysis parameters required for the finite element analysis. Most input data are either available in the literature or may be easily estimated. Product resistance to water vapor flow, mass transfer coefficients describing secondary drying, and container heat transfer coefficients must normally be measured. Each element (i.e., each small subsystem of the product) may be assigned different values of product resistance to accurately describe the nonlinear resistance behavior often shown by real products. During primary drying, the chamber pressure and shelf temperature may be varied in steps. During secondary drying, the change in gas composition from pure water to mostly inert gas is calculated by the model from the instantaneous water vapor flux and the input pumping capacity of the freeze dryer. Results. Comparison of the theoretical results with the experiment data for a 3% sucrose formulation is generally satisfactory. Primary drying times agree within two hours, and the product temperature vs. time curves in primary drying agree within about ± 1°C. The residual moisture vs. time curve is predicted by the theory within the likely experimental error, and the lack of large variation in moisture within the vial (i.e., top vs. side vs. bottom) is also correctly predicted by theory. The theoretical calculations also provide the time variation of “Tg–T” during both primary and secondary drying, where T is product temperature and Tg is the glass transition temperature of the product phase. The calculations demonstrate that with a secondary drying protocol using a rapid ramp of shelf temperature, the product temperature does rise above Tg during early secondary drying, perhaps being a factor in the phenomenon known as “cake shrinkage”. Conclusion. The theoretical results of in-process product temperature, primary drying time, and moisture content mapping and history are consistent with the experimental results, suggesting the theoretical model should be useful in process development and “trouble-shooting” applications.

Structure of carbohydrate-bound polynuclear iron oxyhydroxide nanoparticles in parenteral formulations

Intravenous iron therapy is used to treat anemia associated with chronic kidney disease. The chemical structures of parenteral iron agents have not been characterized in detail, and correlations between structure, efficiency of iron delivery, and toxicity via catalysis of oxygen-derived free radical creation remain to be established. In this study, two formulations of parenteral iron have been characterized by absorption spectroscopy, X-ray diffraction analysis (XRD), transmission electron microscopy (TEM), atomic force microscopy (AFM), and elemental analysis. The samples studied were Venofer ® (Iron Sucrose Injection, USP) and Ferrlecit ® (Sodium Ferric Gluconate in Sucrose Injection). The 250–800-nm absorption spectra and the XRD patterns showed that both formulations contain a mineral core composed of iron oxyhydroxide in the β-FeOOH mineral polymorph known as akaganeite. This was further confirmed for each formulation by imaging using TEM and AFM. The average core size for the nanoparticles, after dialysis to remove unbound or loosely bound carbohydrate, was approximately 3 ± 2 nm for the iron–sucrose, and approximately 2 ± 1 nm for the iron–gluconate. Each of the nanoparticles consists of a mineral core, surrounded by a layer of bound carbohydrate. The overall diameter of the average bead in the dialyzed preparations was approximately 7 ± 4 nm for the iron–sucrose, and 3 ± 1 nm for the iron–gluconate. Undialyzed preparations have particles with larger average sizes, depending on the extent of dilution of unbound and loosely bound carbohydrate. At a dilution corresponding to a final Fe concentration of 5 mg/mL, the average particle diameter in the iron–sucrose formulation was approximately 22 ± 9 nm, whereas that of the iron–gluconate formulation was approximately 12 ± 5 nm.

Determination of iron sucrose (Venofer) or iron dextran (DexFerrum) removal by hemodialysis: an in-vitro study.

BackgroundIntravenous iron is typically administered during the hemodialysis (HD) procedure. HD patients may be prescribed high-flux (HF) or high-efficiency (HE) dialysis membranes. The extent of iron sucrose and iron dextran removal by HD using HF or HE membranes and by ultrafiltration rate (UFR) is unknown.MethodsTwo in vitro HD systems were designed and constructed to determine the dialyzabiltiy of iron from a simulated blood system (SBS) containing 100 mg iron sucrose or iron dextran (system A) or 1000 mg iron sucrose (system B). Both in vitro systems utilized a 6-L closed-loop SBS system that was subject to 4 different HD conditions conducted over 4 hours: HE membrane + 0 ml/hr UFR; HE membrane + 500 ml/hr UFR; HF membrane + 0 ml/hr UFR; HF membrane + 500 ml/hr UFR. Blood flow and dialysate flow rates were 500 ml/min and 800 ml/min, respectively. The dialysate compartment was a 192-L open system for system A and a 6-L closed-loop system for system B. Samples from the SBS and dialysate compartments were taken at various time points and iron elimination rate and HD clearance was determined. Iron removal from the SBS > 15% was considered clinically significant.ResultsThe greatest percentage removal from the SBS was 13.5% and -0.03% utilizing system A and B, respectively. Iron sucrose and iron dextran dialysate concentration was below the lower limits of assay (< 2 ppm) for system A. Dialysate recovery of iron was negligible: 0 – 5.4 mg system A and 5.47 – 23.59 mg for system B. Dialyzer type or UFR did not affect iron removal.ConclusionHF or HE dialysis membranes do not remove clinically significant amounts of iron sucrose or dextran formulations over a 4-hour HD session. This effect remained constant even controlling for UFR up to 500 ml/hour. Therefore, iron sucrose and iron dextran are not dialyzed by HE or HF dialysis membranes irrespective of UFR.

Liposomes and disaccharides as carriers in spray-dried powder formulations of superoxide dismutase

In this study, we aim to investigate the effects of disaccharides and liposome carriers on the activity, solid state characteristics, structural preservation, and aerosol powder performance of spray-dried superoxide dismutase (SOD) formulations. Sucrose, trehalose, and lactose were selected as stabilizing adjuvants in the spray-drying process. Dipalmitoyl phosphatidylcholine (DPPC) was the major lipid component for preparing liposomes. These SOD formulations were characterized with SOD activity assay, particle size, residual moisture content, scanning electron microscopy (SEM), electron spectroscopy for chemical analysis (ESCA), differential scanning calorimeter (DSC), and Fourier transform infrared (FT-IR) spectroscopy. We found that the inlet/outlet temperature of spray drying can be performed up to 168/122 °C with maintaining the activity of SOD in the SOD\\DPPC\\sucrose formulation for 98%. The SEM image of this formulation showed wrinkled and raisin-like appearance. Aerosol powder performance test demonstrated that this formulation exhibited excellent emitted dose (ED, 71%), aerodynamic diameter (2 μm), and respirable fraction (RF, 72%). DSC study suggested an indication of initial electrostatic stabilization of SOD by DPPC and sucrose, the following lipid perturbation by SOD, and the formation of an inclusion complex, thus minimizing the individual transition peaks of SOD and DPPC. FT-IR study showed that the major secondary structure of SOD, β-sheet, was maintained in this formulation. The surface ESCA analysis of this formulation suggested the absence of SOD on the surface region of the powders, indicating that SOD was well surrounded and protected by DPPC and sucrose. Spray drying has been demonstrated to be a feasible process to preserve the activity of SOD in the formulation of DPPC liposomes with sucrose.

A new mechanism for decreasing aggregation of recombinant human interferon‐γ by a surfactant: Slowed dissolution of lyophilized formulations in a solution containing 0.03% polysorbate 20

To study the mechanisms by which Tween 20 (polysorbate 20) used in a reconstitution solution affects the aggregation of lyophilized recombinant human interferon‐γ (rhIFN‐γ), we used four types of buffered formulations containing 0.4–5 mg/mL rhIFN‐γ in either 10 mM potassium phosphate or phosphate buffered saline: (1) without excipients, (2) with 5% sucrose, (3) with 0.03% polysorbate 20, or (4) with the combination of 5% sucrose and 0.03% polysorbate 20. After lyophilization, infrared spectroscopy was used to analyze the secondary structure of the protein in the freeze‐dried solid. Each solid showed structural perturbation of the protein. Each formulation was reconstituted with water or a 0.03% polysorbate 20 solution. Aggregation of rhIFN‐γ after reconstitution was measured by optical density at A350, and recovery of soluble protein was determined by high‐performance liquid chromatography and ultraviolet spectroscopy. After reconstitution with a 0.03% polysorbate 20 solution, aggregation levels in all formulations were either reduced or similar to those found after reconstitution with water. These results revealed the potential for recovery of native protein using the appropriate reconstitution conditions, even though the protein is non‐native in the lyophilized state. Urea‐induced unfolding with and without polysorbate 20 as measured by second‐derivative ultraviolet spectroscopy indicated that a concentration of 0.03% polysorbate 20 lowered the free energy of unfolding for rhIFN‐γ (destabilizing). Polysorbate 20 also retarded refolding from urea solutions and increased aggregation. At a level of 0.03%, polysorbate 20 did not protect the protein against surface‐induced aggregation during agitation. Dissolution times in water versus a 0.03% polysorbate 20 solution were measured using a rotating disk electrode for lyophilized formulations containing an electrochemically reactive species. The presence of 0.03% polysorbate 20 in the reconstitution solution nearly doubled the time required for dissolution of the phosphate buffered saline formulation, and the sucrose formulations dissolved 33–57% more slowly. Slowing the dissolution rates of lyophilized powders allows more time for the protein to refold while it decreases the maximum concentration of the protein at the dissolution interface, thus reducing the total amount of aggregation.

Electrochemical determination of dissolution rates of lyophilized pharmaceutical formulations.

We present a method to measure dissolution times for rapidly dissolving lyophilized formulations. K4Fe(CN)6 was lyophilized in formulations containing sucrose, salts, and Tween 20. Dissolution of the lyophilized powders was measured by monitoring the time dependence of the oxidation of Fe(CN)6(4-) ion at the surface of a platinum rotating disk electrode. Reconstitution of lyophilized K4Fe(CN)6 formulations with aqueous solutions of 0.03% Tween 20 altered the time of dissolution for all cases. Salt and sucrose formulations without Tween 20 dissolved more slowly in a Tween 20 solution than in water alone. In contrast, formulations containing Tween 20 dissolved faster in the Tween 20 solution when compared to dissolution in water.

Development of formulations that enhance physical stability of viral vectors for gene therapy.

This study summarizes our initial efforts to address an issue that is critical to the success of any multicenter gene therapy clinical trial - maintenance of vector viability during shipping and storage at remote test sites. We have identified formulation and processing factors that influence stability of viral preparations such as selection of appropriate buffer systems, cryoprotectants, and storage conditions. Adenovirus and adeno-associated virus expressing E. coli beta-galactosidase (lacZ) were suspended in blends of complex carbohydrates, cyclodextrins and various surfactants. X-gal stains of 293 and 84-31 cells were used to determine infectious titer of all preparations. Potassium phosphate-buffered preparations consistently maintained high viral titers after storage at -20 and 4 degrees C. Blends of sucrose, mannitol, and surfactant showed negligible loss of titer for 35 days at 4 degrees C. Formulations of sucrose and cyclodextrin were stable for 2 years at -20 degrees C. Negligible loss in titer was observed in unit-dose viral preparations lyophilized in sucrose and stored at 4 degrees C for 1 year after an initial loss of 0.5 log due to processing. Studies with lyophilized sucrose/mannitol blends have shown that viral recovery after processing is directly related to the final moisture content of the dried product. Virus concentration also plays a significant role in recovery after processing with highly concentrated preparations showing minimal loss in titer after lyophilization. In summary, lyophilized preparations that can be shipped and stored at 25 degrees C offer a solution to the current problem of distribution of viral vectors for clinical trials.

Production processes of licensed recombinant factor VIII preparations.

The state-of-the-art treatment for hemophilia A is replacement therapy with recombinant factor VIII (rFVIII) made possible by genetic engineering advances. Currently, there are four different products licensed and available for hemophilia A patients. All are produced by recombinant mammalian cells in large-scale fermenter cultures, purified to high purity, formulated in stable formulations and freeze dried. The first-generation products Recombinate and Kogenate (also sold as Helixate by Aventis) are characterized as full-length human factor VIII molecules and formulated using human serum albumin as a stabilizer. The second-generation product ReFacto contains an improved albumin-free sucrose formulation and incorporates advanced antiviral safety procedures in the manufacturing process. It is a truncated B region-deleted form of factor VIII (FVIII) that makes use of a nonhuman peptide linker 14 amino acids in length to connect the 80 and 90 kD subunits. The most recently licensed rFVIII product is the second-generation Kogenate product called KOGENATE Bayer/Kogenate FS, which combines the advantages of the human full-length FVIII molecule with an albumin-free, sucrose-based synthetic formulation as well as an improved viral safety profile. In this article, the manufacturing processes for each of the four different products are discussed in detail, focusing on expression systems and cell lines, culture medium, technical culture systems, purification process (including viral removal potential), and final formulation.

Optimization of Storage Stability of Lyophilized Actin Using Combinations of Disaccharides and Dextran

The storage stability of a dry protein depends on the structure of the dried protein, as well as on the storage temperature relative to the glass transition temperature of the dried preparation. Disaccharides are known to preserve the native conformation of a dried protein; however, the resulting Tg of the sample may be too low ensure adequate storage stability. On the other hand, formulations dried with high molecular weight carbohydrates, such as dextran, have higher glass transition temperatures, but fail to preserve native protein conformation. We tested the hypothesis that optimizing both protein structure and Tg by freeze‐drying actin with mixtures of disaccharides and dextran would result in increased storage stability compared to actin dried with either disaccharide or dextran alone. Protein structure in the dried solid was analyzed immediately after lyophilization and after storage at elevated temperatures with infrared spectroscopy, and after rehydration by infrared and circular dichroism spectroscopy. Structural results were related to the polymerization activity recovered after rehydration. Degradation was noted with storage for formulations containing either sucrose, trehalose, or dextran alone. Slight increases in Tg observed in trehalose formulations compared to sucrose formulations did not result in appreciable increases in storage stability. Addition of dextran to sucrose or trehalose increased formulation Tg without affecting the capacity of the sugar to inhibit protein unfolding during lyophilization and resulted in improved storage stability. Also, dextran provides an excellent amorphous bulking agent, which can be lyophilized rapidly with formation of strong, elegant cake structure. These results suggest that the strategy of using a mixture of disaccharide and polymeric carbohydrates can optimize protein storage stability. © 2000 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89: 199–214, 2000

The relationship between protein aggregation and molecular mobility below the glass transition temperature of lyophilized formulations containing a monoclonal antibody.

To find out if the physical instability of a lyophilized dosage form is related to molecular mobility below the glass transition temperature. Further, to explore if the stability data generated at temperatures below the glass transition temperature can be used to predict the stability of a lyophilized solid under recommended storage conditions. The temperature dependence of relaxation time constant, tau, was obtained for sucrose and trehalose formulations of the monoclonal antibody (5 mg protein/vial) from enthalpy relaxation studies using differential scanning calorimetry. The non-exponentiality parameter, beta, in the relaxation behavior was also obtained using dielectric relaxation spectroscopy. For both sucrose and trehalose formulations, the variation in tau with temperature could be fitted Vogel-Tammann-Fulcher (VTF) equation. The two formulations exhibited difference sensitivities to temperature. Sucrose formulation was more fragile and exhibited a stronger non-Arrhenius behavior compared to trehalose formulation below glass transition. Both formulations exhibited < 2% aggregation at t/tau values < 10, where t is the time of storage. Since the relaxation times for sucrose and trehalose formulations at 5 degrees C are on the order of 10(8) and 10(6) hrs, it is likely that both formulations would undergo very little (< 2%) aggregation in a practical time scale under refrigerated conditions.

Effect of glass transition temperature on the stability of lyophilized formulations containing a chimeric therapeutic monoclonal antibody.

The purpose of this study is to highlight the importance of knowing the glass transition temperature, Tg, of a lyophilized amorphous solid composed primarily of a sugar and a protein in the interpretation of accelerated stability data. Glass transition temperatures were measured using DSC and dielectric relaxation spectroscopy. Aggregation of protein in the solid state was monitored using size-exclusion chromatography. Sucrose formulation (Tg approximately 59 degrees C) when stored at 60 degrees C was found to undergo significant aggregation, while the trehalose formulation (Tg approximately 80 degrees C) was stable at 60 degrees C. The instability observed with sucrose formulation at 60 degrees C can be attributed to its Tg (approximately 59 degrees C) being close to the testing temperature. Increase in the protein/sugar ratio was found to increase the Tgs of the formulations containing sucrose or trehalose, but to different degrees. Since the formulations exist in glassy state during their shelf-life, accelerated stability data generated in the glassy state (40 degrees C) is perhaps a better predictor of the relative stability of formulations than the data generated at a higher temperature (60 degrees C) where one formulation is in the glassy state while the other is near or above its Tg.

Comparative Toxicity of Aqueous Solutions of Boric Acid and Polybor 3 to House Flies (Diptera: Muscidae)

LC50s and LT50s of boric acid and polybor formulated in water and 10% sucrose were determined for 3- to 5-d-old adult house flies, Musca domestica (L.), of mixed sex. Differences between boric acid and polybor toxicities were significant in 10% sucrose, but not in water. However, borates formulated in water had significantly lower LC50s than those formulated in 10% sucrose. Rate of kill for formulations in water was fairly uniform over time, whereas mortality from sucrose formulations was not observed until 17 h after treatment. Reasons for differences in the manifestation of mortality and possibilities for practical application are discussed.

Laboratory Studies of Viral Adjuvants: Formulation Development1

Addition of soybean, corn, or cottonseed components and sugars to water-based formulations containing Baculovirus heliothis , the nuclear polyhedrosis virus of Heliothis sp., produced significantly higher mortalities in laboratory tests against 24-hold bollworm, Heliothis zea (Boddie), larvae than did a standard, water-based virus formulation. Formulations containing soybean flour and soybean oil or corn flour and corn oil produced significantly greater bollworm mortalittes than formulations containing only the corresponding oil or flour. Mortalities were not significantly different among citrus pulp, cottonseed, and soybean adjuvant formulations. Bollworm mortality decreased as the percentage of sucrose was increased in increments from 5 to 20% in the soybean flour + soybean oil (SFO) formulation. Substitution of lactose, maltose, or glucose for sucrose in the SFO formulation did not increase mortality but increased costs significantly. Addition of a polysaccharide thickener or ultraviolet (UV) protectant to the SFO + sucrose formulation did not significantly reduce bollworm mortality. The SFO + sucrose formulation was found to be equally effective when volumetric application rates or virus concentrations were varied. The ratio of mortalities caused by adjuvant suspensions containing combinations of soybean flour, soybean oil, sucrose, virus, and water to mortalities caused by virus-water suspension alone usually varied from 6.4 to 15, although one ratio was 55. Estimated cost of the SFO adjuvant (based on current prices, September 1979) at an application rate of 28 liters/ha would be $1.66/ha when an SFO concentration of 2.66 kg/28 liters, or 9.5% (wt/vol), is used.