Solution stability of Captisol-stabilized melphalan (Evomela) versus Propylene glycol-based melphalan hydrochloride injection.

PURPOSE: The objective of this study was to compare the stability of recently approved Captisol-stabilized propylene glycol-free melphalan injection (Evomela™) against currently marketed propylene glycol-based melphalan injection. The products were compared as reconstituted solutions in vials as well as admixture solutions prepared from normal saline in infusion bags. METHODS: Evomela and propylene glycol-based melphalan injection were reconstituted in normal saline and organic custom diluent, respectively, according to their package insert instructions. The reconstituted solutions were diluted in normal saline to obtain drug admixture solutions at specific drug concentrations. Stability of the solutions was studied at room temperature by assay of melphalan and determination of melphalan-related impurities. RESULTS: Results show that based on the increase in total impurities in propylene glycol-based melphalan injection at 0.45 mg/mL, Evomela admixture solutions are about 5, 9, 15 and 29 times more stable at concentrations of 0.45, 1.0, 2.0 and 5.0 mg/mL, respectively. Results confirmed that reconstituted Evomela solution can be stored in the vial for up to 1 h at RT or for up to 24 h at refrigerated temperature (2-8 °C) with no significant degradation. After storage in the vial, it remains stable for an additional 3-29 h after preparation of admixture solution in infusion bags at concentrations of 0.25-5.0 mg/mL, respectively. In addition, Evomela solution in saline, at concentration of 5.0 mg/mL melphalan was bacteriostatic through 72 h storage at 2-8 °C. CONCLUSION: Formulation of melphalan with Captisol technology significantly improved stability compared to melphalan hydrochloride reconstituted with propylene-glycol based diluents.

Development and validation of a rapid ultra-high performance liquid chromatography method for the assay of benzalkonium chloride using a quality-by-design approach.

A rapid robust reversed-phase UHPLC method has been developed for the analysis of total benzalkonium chloride in preserved drug formulation. A systematic Quality-by-Design (QbD) method development approach using commercial, off the shelf software (Fusion AE(®)) has been used to optimize the column, mobile phases, gradient time, and other HPLC conditions. Total benzalkonium chloride analysis involves simple sample preparation. The method uses gradient elution from an ACE Excel 2 C18-AR column (50mm×2.1mm, 2.0µm particle size), ammonium phosphate buffer (pH 3.3; 10mM) as aqueous mobile phase and methanol/acetonitrile (85/15, v/v) as the organic mobile phase with UV detection at 214nm. Using these conditions, major homologs of the benzalkonium chloride (C12 and C14) have been separated in less than 2.0min. The validation results confirmed that the method is precise, accurate and linear at concentrations ranging from 0.025mg/mL to 0.075mg/mL for total benzalkonium chloride. The recoveries ranged from 99% to 103% at concentrations from 0.025mg/mL to 0.075mg/mL for total benzalkonium chloride. The validation results also confirmed the robustness of the method as predicted by Fusion AE(®).

Analysis of drug-protein binding by ultrafast affinity chromatography using immobilized human serum albumin.

Human serum albumin (HSA) was explored for use as a stationary phase and ligand in affinity microcolumns for the ultrafast extraction of free drug fractions and the use of this information for the analysis of drug-protein binding. Warfarin, imipramine, and ibuprofen were used as model analytes in this study. It was found that greater than 95% extraction of all these drugs could be achieved in as little as 250 ms on HSA microcolumns. The retained drug fraction was then eluted from the same column under isocratic conditions, giving elution in less than 40 s when working at 4.5 mL/min. The chromatographic behavior of this system gave a good fit with that predicted by computer simulations based on a reversible, saturable model for the binding of an injected drug with immobilized HSA. The free fractions measured by this method were found to be comparable to those determined by ultrafiltration, and equilibrium constants estimated by this approach gave good agreement with literature values. Advantages of this method include its speed and the relatively low cost of microcolumns that contain HSA. The ability of HSA to bind many types of drugs also creates the possibility of using the same affinity microcolumn to study and measure the free fractions for a variety of pharmaceutical agents. These properties make this technique appealing for use in drug-binding studies and in the high-throughput screening of new drug candidates.

Studies of verapamil binding to human serum albumin by high-performance affinity chromatography.

The binding of verapamil to the protein human serum albumin (HSA) was examined by using high-performance affinity chromatography. Many previous reports have investigated the binding of verapamil with HSA, but the exact strength and nature of this interaction (e.g. the number and location of binding sites) is still unclear. In this study, frontal analysis indicated that at least one major binding site was present for R- and S-verapamil on HSA, with estimated association equilibrium constants on the order of 10(4)M(-1) and a 1.4-fold difference in these values for the verapamil enantiomers at pH 7.4 and 37 degrees C. The presence of a second, weaker group of binding sites on HSA was also suggested by these results. Competitive binding studies using zonal elution were carried out between verapamil and various probe compounds that have known interactions with several major and minor sites on HSA. R/S-Verapamil was found to have direct competition with S-warfarin, indicating that verapamil was binding to Sudlow site I (i.e. the warfarin-azapropazone site of HSA). The average association equilibrium constant for R- and S-verapamil at this site was 1.4 (+/-0.1)x10(4)M(-1). Verapamil did not have any notable binding to Sudlow site II of HSA but did appear to have some weak allosteric interactions with l-tryptophan, a probe for this site. An allosteric interaction between verapamil and tamoxifen (a probe for the tamoxifen site) was also noted, which was consistent with the binding of verapamil at Sudlow site I. No interaction was seen between verapamil and digitoxin, a probe for the digitoxin site of HSA. These results gave good agreement with previous observations made in the literature and help provide a more detailed description of how verapamil is transported in blood and of how it may interact with other drugs in the body.

Development of immunoaffinity restricted access media for rapid extractions of low-mass analytes.

Restricted access media using antibodies as immobilized ligands were developed for the rapid and selective capture of small analytes by immunoextraction, giving rise to materials referred to as immunoaffinity restricted access media (IA-RAM). To make such a material, intact antibodies for the desired target were first immobilized onto porous silica, with antibodies at or near the outer surface of the support then being treated with papain (or a related agent) to release and remove their binding domains. The result was a support in which only antibodies deep within the pores remained intact and able to bind to the target. Items evaluated in the development of such media included the immobilization method used for the antibodies, the pore size of the support, and the amount of papain and time that were used for support treatment. A theoretical model was also developed to describe the extent of binding domain removal based on the measured polypeptide content of the IA-RAM support before and after treatment with papain. The final optimized conditions for making the IA-RAM supports were used to prepare columns that contained antifluorescein antibodies. Injections of fluorescein and fluorescein-labeled bovine serum albumin onto these IA-RAM columns gave selective and quantitative extraction of fluorescein in 1-2 s. This approach can be used with other antibodies and low-mass targets and should be valuable for such applications as the rapid separation of drugs from drug-protein complexes or the isolation of labeled/modified peptides from intact proteins that contain the same modification or label.

Immobilization of alpha1-acid glycoprotein for chromatographic studies of drug-protein binding II. correction for errors in association constant measurements.

A new method for the immobilization of alpha(1)-acid glycoprotein (AGP) in high-performance liquid chromatography (HPLC) columns was recently described for applications such as drug binding studies. Part of this earlier work used self-competition zonal elution studies to measure association equilibrium constants between immobilized AGP and R- or S-propranolol. It was later found that analysis of these data by a common equation derived for linear elution conditions gave erroneous values for experiments actually conducted under nonlinear conditions. This report discusses the nature of this error and uses frontal analysis to estimate the true binding strength between R- and S-propranolol and HPLC columns containing immobilized AGP.

Development of an affinity silica monolith containing human serum albumin for chiral separations.

An affinity monolith based on silica and containing immobilized human serum albumin (HSA) was developed and evaluated in terms of its binding, efficiency and selectivity in chiral separations. The results were compared with data obtained for the same protein when used as a chiral stationary phase with HPLC-grade silica particles or a monolith based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA). The surface coverage of HSA in the silica monolith was similar to values obtained with silica particles and a GMA/EDMA monolith. However, the higher surface area of the silica monolith gave a material that contained 1.3-2.2-times more immobilized HSA per unit volume when compared to silica particles or a GMA/EDMA monolith. The retention, efficiency and resolving power of the HSA silica monolith were evaluated using two chiral analytes: d/l-tryptophan and R/S-warfarin. The separation of R- and S-ibuprofen was also considered. The HSA silica monolith gave higher retention and higher or comparable resolution and efficiency when compared with HSA columns that contained silica particles or a GMA/EDMA monolith. The silica monolith also gave lower back pressures and separation impedances than these other materials. It was concluded that silica monoliths can be valuable alternatives to silica particles or GMA/EDMA monoliths when used with immobilized HSA as a chiral stationary phase.

Development of an affinity silica monolith containing alpha1-acid glycoprotein for chiral separations.

An affinity monolith based on silica and containing immobilized alpha(1)-acid glycoprotein (AGP) was developed and evaluated in terms of its binding, efficiency and selectivity in chiral separations. The results were compared with data obtained for the same protein when used as a chiral stationary phase with HPLC-grade silica particles or monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA). The surface coverage of AGP in the silica monolith was 18% higher than that obtained with silica particles and 61% higher than that measured for a GMA/EDMA monolith. The higher surface area of the silica monolith gave materials that contained 1.5- to 3.6-times more immobilized protein per unit volume when compared to silica particles or a GMA/EDMA monolith. The retention, efficiency and resolving power of the AGP silica monolith were evaluated by injecting two chiral analytes onto this column (i.e., R/S-warfarin and R/S-propranolol). In each case, the AGP silica monolith gave higher retention plus better resolution and efficiency than AGP columns containing silica particles or a GMA/EDMA monolith. The AGP silica monolith also gave lower back pressures and separation impedances than these other materials. It was concluded that silica monoliths can be valuable alternatives to silica particles or GMA/EDMA monoliths when used with AGP as a chiral stationary phase.

Development of sulfhydryl-reactive silica for protein immobilization in high-performance affinity chromatography.

Two techniques were developed for the immobilization of proteins and other ligands to silica through sulfhydryl groups. These methods made use of maleimide-activated silica (the SMCC method) or iodoacetyl-activated silica (the SIA method). The resulting supports were tested for use in high-performance affinity chromatography by employing human serum albumin (HSA) as a model protein. Studies with normal and iodoacetamide-modified HSA indicated that these methods had a high selectivity for sulfhydryl groups on this protein, which accounted for the coupling of 77-81% of this protein to maleimide- or iodoacetyl-activated silica. These supports were also evaluated in terms of their total protein content, binding capacity, specific activity, nonspecific binding, stability, and chiral selectivity for several test solutes. HSA columns prepared using maleimide-activated silica gave the best overall results for these properties when compared to HSA that had been immobilized to silica through the Schiff base method (i.e., an amine-based coupling technique). A key advantage of the supports developed in this work is that they offer the potential of giving greater site-selective immobilization and ligand activity than amine-based coupling methods. These features make these supports attractive in the development of protein columns for such applications as the study of biological interactions and chiral separations.

Affinity monolith chromatography.

The combined use of monolithic supports with selective affinity ligands as stationary phases has recently given rise to a new method known as affinity monolith chromatography (AMC). This review will discuss the basic principles behind AMC and examine the types of supports and ligands that have been employed in this method. Approaches for placing affinity ligands in monoliths will be considered, including methods based on covalent immobilization, biospecific adsorption, entrapment, and the formation of coordination complexes. Several reported applications will then be presented, such as the use of AMC for bioaffinity chromatography, immunoaffinity chromatography, immobilized metal-ion affinity chromatography, dye-ligand affinity chromatography, and biomimetic chromatography. Other applications that will be discussed are chiral separations and studies of biological interactions based on AMC.

Applications of silica supports in affinity chromatography.

The combined use of silica-based chromatographic supports with immobilized affinity ligands can be used in many preparative and analytical applications. One example is the use of silica-based affinity columns in HPLC, giving rise to a method known as high-performance affinity chromatography (HPAC). This review discusses the role that silica has played in the development of affinity chromatography and HPAC and the applications of silica in these methods. This includes a discussion of the types of ligands that have been employed with silica and the methods by which these ligands have been immobilized. Various formats have also been presented for the use of silica in affinity chromatographic methods, including assays involving direct or indirect analyte detection, on-line or off-line affinity extraction, and chiral separations. The use of silica-based affinity columns in studies of biological systems based on zonal elution and frontal analysis methods will also be considered.

Chromatographic analysis of carbamazepine binding to human serum albumin. II. Comparison of the Schiff base and N-hydroxysuccinimide immobilization methods.

Recent studies with carbamazepine on human serum albumin (HSA) columns have noted an appreciable degree of non-specific binding on supports prepared by the Schiff base immobilization method. This work examines an alternative immobilization method for HSA based on N-hydroxysuccinimide (NHS)-activated silica. This support was prepared by reacting HPLC-grade silica directly with disuccinimidyl carbonate. The resulting material was compared to an HSA support prepared by the Schiff base method in terms of its activity for carbamazepine and non-specific interactions with this drug. When examined by frontal analysis, both supports gave comparable association equilibrium constants for carbamazepine interactions with HSA ((0.53-0.55) x 10(4)M(-1) at 37 degrees C). However, columns prepared by the Schiff base method gave greater non-specific binding. These columns, as well as control columns prepared using the carbonyldiimidazole (CDI) immobilization method, were also evaluated for their non-specific binding to a variety of other solutes known to interact with HSA. From these results it was concluded that the NHS method was an attractive alternative to the Schiff base technique in the preparation of immobilized HSA for HPLC-based binding studies for carbamazepine. However, it was also noted that non-specific binding varies from one drug to the next in these immobilization methods, indicating that such properties should be evaluated on a case-by-case basis in the use and development of HSA columns for binding studies.

Affinity monoliths for ultrafast immunoextraction.

Affinity monoliths based on a copolymer of glycidyl methacrylate and ethylene dimethacrylate were developed for ultrafast immunoextractions. Rabbit immunoglobulin G (IgG) and anti-FITC antibodies were used as model ligands for this work. The antibody content of the monoliths was optimized by varying both the polymerization and immobilization conditions for preparing such supports. The temperature and porogen composition used during polymerization showed significant effects on monolith morphology and on the amount of antibodies that could be coupled to these materials. The effects of various immobilization procedures and coupling conditions were also evaluated, including the coupling temperature, pH, protein concentration, and use of high buffer concentrations. The maximum ligand density obtained for rabbit IgG was approximately 60 mg/g. When a 4.5 mm i.d. x 0.95 mm monolith disk containing anti-FITC antibodies was used, 95% extraction of fluorescein was achieved in 100 ms. These properties make such monoliths attractive for work in the rapid isolation of analytes from biological samples. Similar columns can be developed for other targets by varying the types of antibodies or binding agents placed within the monoliths.

High-performance affinity monolith chromatography: development and evaluation of human serum albumin columns.

Several immobilization methods were explored for the preparation of high-performance affinity monolithic columns containing human serum albumin (HSA). These monoliths were based on a copolymer of glycidyl methacrylate and ethylene dimethacrylate. In one method, the epoxy groups of this copolymer were used directly for the immobilization of HSA through its amine residues (i.e., the epoxy method); in other approaches, these epoxy groups were converted to diols for later use in the carbonyldiimidazole, disuccinimidyl carbonate, and Schiff base methods. Each HSA monolith was evaluated in terms of its total protein content and its retention of several model compounds, including (R/S)-warfarin and D/L-tryptophan. The greatest amount of immobilized HSA was obtained by the Schiff base method, whereas the epoxy method gave the lowest protein content. The Schiff base method also gave the best resolution in chiral separations of (R/S)-warfarin and D/L-tryptophan. All of the immobilization methods gave similar relative activities for HSA in its binding to (R)- and (S)-warfarin, but some differences were noted in the activity of the immobilized HSA for D- and L-tryptophan. The efficiency of these monoliths was found to be greater than that of silica-based HSA columns for (R/S)-warfarin (i.e., analytes with high retention), but little or no difference was seen for D- and L-tryptophan (analytes with weak retention).