Development of separation technology for the removal of radium-223 from targeted thorium conjugate formulations. Part II: purification of targeted thorium conjugates on cation exchange columns.

Tumor targeting pharmaceuticals will play a crucial role in future pharma pipelines. The targeted thorium conjugate (TTC) therapeutic platform could provide real benefit to patients, whereby targeting moieties like monoclonal antibodies are radiolabelled with the alpha-emitting radionuclide thorium-227 (227Th, t1/2 = 18.7 days). A potential problem could be the accumulation of the long-lived daughter nuclide radium-223 (223Ra, t1/2 = 11.4 days) in the drug product during manufacturing and distribution. Therefore, the level of 223Ra must be standardized before administration to the patient. The focus in this study has been the removal of 223Ra, as the other progenies will have a very limited stay in the formulation. In this study, the purification of TTCs labeled with decayed 227Th has been explored. Columns packed with a strong cation exchange resin have been used to sequester 223Ra. The separation of TTC from 223Ra has been evaluated as influenced by both formulation and process parameters with a design of experiments (DOE) study; including citrate or acetate buffer, pH, buffer concentration, presence or absence of pABA + EDTA, resin amount and sodium chloride concentration. The aim was to achieve a separation with high sorption of 223Ra and accompanying low TTC sorption. The results were analyzed by multivariate analysis. Four regression models of TTC and 223Ra sorption from citrate and acetate buffered formulations were developed. The predictive accuracy of sorption in the four statistical models was given by standard deviations and confidence intervals. The TTC sorption in citrate and acetate buffered formulations was affected by the identical variables and the variation in TTC sorption was comparable for the two models. However, the DOE variables had a significantly stronger impact on the 223Ra sorption in citrate buffered formulations than the 223Ra sorption in acetate buffer. An optimal separation with a TTC sorption below 25% and 223Ra sorption above 90% can be achieved in both citrate and acetate buffered formulations. Stability studies of radiochemical purity (RCP) indicated that the measured 227Th values may be partly due to free 227Th and not TTC, but the results indicate that TTC stability may be controlled by optimizing formulation parameters. Hence, the sorption data and the regression models presented must be reviewed and further explored with regard to what is known about the stability of the TTC in the different buffered formulations.

Development of separation technology for the removal of radium-223 from targeted thorium conjugate formulations. Part I: purification of decayed thorium-227 on cation exchange columns.

Targeted thorium conjugates (TTCs) are being explored as a potential future platform for specific tumor targeting pharmaceuticals. In TTCs, the alpha emitting radionuclide thorium-227 (227Th) with a half-life of 18.697 d is labeled to targeting moieties, such as monoclonal antibodies (mAbs). The amount of daughter nuclide radium-223 (223Ra, t1/2 = 11.435 d) will increase during manufacture and distribution, and so a technology for purification is required to assure an acceptable level of 223Ra is administrated to the patient. Since 223Ra is the only progeny of 227Th with a long half-life (days), the progenies of 223Ra will have a very limited stay in the formulation once 223Ra is removed. The focus in this study has, therefore, been on the removal of 223Ra. In this study, the sorption and separation of 223Ra (radium(II)) and 227Th (thorium(IV)) on cation exchange columns has been evaluated as a purification method of decayed 227Th (i.e. prior to radiolabelling of a mAb and formation of TTC). The goal is to minimize the sorption of 227Th and maximize the sorption of 223Ra. Statistical experimental design with formulation and process parameters, including buffered formulations comprising citrate and acetate, at various concentrations and pH, presence of free radical scavenger and chelator, and resin amount have been evaluated for impact on the purification process. The studies have been interpreted by the aid of multivariate data analysis. The correlations between design of experimental variables and sorption are summarized by regression models. The predictive accuracy of radionuclide sorption was given by standard deviation and 95% confidence intervals originating from statistical cross validation. Experimental results and statistical models for citrate-buffered formulations verified reproducible and acceptable sorption levels of 223Ra and 227Th under selected conditions. For acetate-buffered formulations, prediction of 227Th sorption was influenced by complex variable relationships and hence a risk of obtaining irreproducibility. Fine-tuned variable levels showed, however, variable combinations predicting high sorption of 223Ra (>90%) and low sorption of 227Th (<3%) also for the acetate-buffered formulations. The optimal separation conditions should be decided based on tuning the variables levels for 223Ra in the citrate-buffered formulations, while for acetate, the optimal separation should be based on tuning variable levels for 227Th sorption. The ionic strength of the formulation also seemed to affect the radionuclide sorption. Labeling of an antibody-chelator conjugate with purified 227Th (i.e. preparation of TTC) was successful in the selected citrate-buffered formulations tested.

The influence of polysorbate 80 on the radiochemical synthesis of a PET tracer in the FASTlab.

PURPOSE: The aim of current study was to investigate the influence of a common non-ionic surfactant, polysorbate 80 (PS80), on radioactive labelling process of a novel PET tracer, [(18)F]Flutemetamol. METHODS: Ferrous oxidation-xylenol orange (FOX) assay, in addition to UV/VIS and (1)H NMR spectroscopies were applied to characterise the composition of the PS80 solution after storage. Multivariate Curve Resolution (MCR) and PLS analysis was used to establish correlation between quality of the PS80 solution and the RCP obtained after labelling. RESULTS: The levels of unsaturated fatty acid moieties of PS80 were negatively correlated to RCP of [(18)F]Flutemetamol after synthesis. This explains the slight increase in RCP when stored PS80 solutions were applied in the synthesis. The mechanism behind this observation is suggested to be related to radiation induced radical formation in the unsaturated fatty acids, which subsequently causes instability of the PET tracer. UV/VIS spectroscopy was demonstrated to have the ability as a possible control tool for quality assurance of the studied radioactive labelling process. CONCLUSIONS: The presence of unsaturated fatty acid moieties in PS80 was found to be one of the most important factors responsible for the reduction in RCP of [(18)F]Flutemetamol after synthesis.

Classification of structurally related commercial contrast media by near infrared spectroscopy.

Near infrared spectroscopy (NIRS) is a non-destructive measurement technique with broad application in pharmaceutical industry. Correct identification of pharmaceutical ingredients is an important task for quality control. Failure in this step can result in several adverse consequences, varied from economic loss to negative impact on patient safety. We have compared different methods in classification of a set of commercially available structurally related contrast media, Iodixanol (Visipaque(®)), Iohexol (Omnipaque(®)), Caldiamide Sodium and Gadodiamide (Omniscan(®)), by using NIR spectroscopy. The performance of classification models developed by soft independent modelling of class analogy (SIMCA), partial least squares discriminant analysis (PLS-DA) and Main and Interactions of Individual Principal Components Regression (MIPCR) were compared. Different variable selection methods were applied to optimize the classification models. Models developed by backward variable elimination partial least squares regression (BVE-PLS) and MIPCR were found to be most effective for classification of the set of contrast media. Below 1.5% of samples from the independent test set were not recognized by the BVE-PLS and MIPCR models, compared to up to 15% when models developed by other techniques were applied.

Strategies for multivariate modeling of moisture content in freeze-dried mannitol-containing products by near-infrared spectroscopy.

Accurate determination of residual moisture content of a freeze-dried (FD) pharmaceutical product is critical for prediction of its quality. Near-infrared (NIR) spectroscopy is a fast and non-invasive method routinely used for quantification of moisture. However, several physicochemical properties of the FD product may interfere with absorption bands related to the water content. A commonly used stabilizer and bulking agent in FD known for variation in physicochemical properties, is mannitol. To minimize this physicochemical interference, different approaches for multivariate correlation between NIR spectra of a FD product containing mannitol and the corresponding moisture content measured by Karl Fischer (KF) titration have been investigated. A novel method, MIPCR (Main and Interactions of Individual Principal Components Regression), was found to have significantly increased predictive ability of moisture content compared to a traditional PLS approach. The philosophy behind the MIPCR is that the interference from a variety of particle and morphology attributes has interactive effects on the water related absorption bands. The transformation of original wavelength variables to orthogonal scores gives a new set of variables (scores) without covariance structure, and the possibility of inclusion of interaction terms in the further modeling. The residual moisture content of the FD product investigated is in the range from 0.7% to 2.6%. The mean errors of cross validated prediction of models developed in the investigated NIR regions were reduced from a range of 24.1-27.6% for traditional PLS method to 15.7-20.5% for the MIPCR method. Improved model quality by application of MIPCR, without the need for inclusion of a large number of calibration samples, might increase the use of NIR in early phase product development, where availability of calibration samples is often limited.

Characterization of polysorbate 80 with liquid chromatography mass spectrometry and nuclear magnetic resonance spectroscopy: specific determination of oxidation products of thermally oxidized polysorbate 80.

The polysorbate species in polysorbate 80 (PS 80) have been characterized with (1)H NMR, LC-UV/MS and MS/MS. The MS was operated with both negative and positive electrospray ionization (ESI). PS 80 was found to contain mono- and di-fatty acid esters of polyoxyethylene (POE) sorbitan, POE isosorbide and of POE. In addition, the same species were also present without fatty acid esters. The polysorbate species were esterified with C12:0, C14:0, C16:1, C16:0, C18:2, C18:1 and C18:0 fatty acids. The main species were polysorbate esters of C18:1. The C18:2 polysorbate species were shown, by (1)H NMR, to have a large component of fatty acids with a conjugated double bond of ZE and/or EZ configuration. The positive ESI mass spectra of the polysorbate species displayed a specific in-source fatty acid fragment for each fatty acid ester. The mass chromatograms of the in-source fatty acid fragments were used to determine the degradation of specific polysorbate species in PS 80. The C18:2 polysorbate species were completely degraded after 8 weeks at 40°C under an atmosphere of air, while the main C18:1 polysorbate species were reduced to ca. 80-86% accompanied by an increase of short-chain POE C18:1 species. Polysorbate species esterified with C18:1-OH, C18:1 keto and C18:0 epoxy acids were found as degradation products of PS 80 stored at 40°C for 8 weeks under air. C18:1-OH, C18:1 keto and C18:0 epoxy acids were believed to be oxidation products of C18:1. With the present conditions, the positive ESI mass spectra of C18:1-OH and C18:0 epoxy polysorbate species displayed identical ions to the C18:2 polysorbate species due to a facile in-source loss of H(2)O from the protonated molecules. The presence of polysorbate esters of C18:1-OH and C18:0 epoxy acids were established using negative ESI MS. The presence of oxidized fatty acids in degraded PS 80 was also confirmed by saponification and extraction followed by negative ESI LC-MS analysis of the free fatty acids.

Stabilization of Gas Bubbles Released from Water-Soluble Carbohydrates Using Amphiphilic Compounds: Preparation of Formulations and Acoustic Monitoring of Bubble Lifetime.

The ultrasound contrast agents Echovist(®) and Levovist(®) (Bayer AG, Schering AG, Germany) are based on the release of gas bubbles from milled α-d-galactose. In diagnostic ultrasound, for this class of contrast agents, there is a need for prolonged contrast duration. To investigate if new carbohydrate compositions could prolong the lifetime of the gas bubbles, α-d-galactose was mixed with other carbohydrates or amphiphiles with varying log P. Acoustic attenuation vs. time (390 s) area under the curve (A(390)) and bubble half-time (t½) were used as measures of prolonged lifetime of gas bubbles. The products, to which 0.1% of a lipophilic carboxylic acid (5ß-cholanic acid, behenic acid, and melissic acid) has been added, showed more than 5, 7 and 11 times enhancement of A(390), respectively, compared with the reference compound 2 (RC2) corresponding to the commercial product Levovist®. The half-time t ½ of the same compounds was prolonged more than 6 times compared with RC2. A partial least square (PLS) statistical analysis confirmed that, for additives, high log P carboxylic acids lead to the highest A(390). The present results bear a promise of products with a more persistent in vivo ultrasound contrast effect than the commercially available agents.

Tuning the MR properties of blood-stable pH-responsive paramagnetic liposomes.

Paramagnetic pH-responsive liposomes have recently been suggested as a promising approach for monitoring by magnetic resonance imaging (MRI) pH changes in tumours. In the present study, the effects of variations in bilayer composition on the relaxometric properties of diacylphosphatidylethanolamine (PE)/dipalmitoylglycerosuccinate (DPSG) liposomal GdDTPA-BMA were investigated both in buffer and blood. A factorial experimental design was used with the variables PE chain length and mol% DPSG. All the relaxometric profiles displayed a semi-sigmoidal shape with a minimum plateau at high pH (r1(min)) and a maximum at low pH (r1(max,E)). Relevant sigmoidal curve fit parameters were evaluated by partial least squares regression. Systematic variations in the relaxometric response (r1(max,E)-r1(min)) were shown for the liposomal systems both in buffer and blood. The pH value at which the r1 was 20% of r1(max,E) relative to r1(min), i.e. pH20, decreased significantly both in buffer and blood as a function of the mol% DPSG. This phenomenon could be understood by the increased surface charge density with increasing mol% DPSG and, hence, higher barrier against liposome aggregation with consequent leakage of contrast agent. Furthermore, the pH relaxometric profiles in blood were shifted laterally to higher, and likely more clinically relevant pH values than the corresponding profiles in buffer. The liposome formulations displayed minimal leakage of contrast agent after prolonged incubation in blood at physiological pH and retained their pH sensitivity after pre-incubation in blood.

Quantum chemical descriptors in the formulation of pectin pellets produced by extrusion/spheronisation.

The objective of this study was to employ quantitative structure-activity relationships (QSAR) to relate calculated molecular descriptors of granulation liquid additives to improvements in the size of pectin pellets produced by extrusion/spheronisation. Quantum chemical descriptors were calculated for a large number of candidate additives. Based on a principal component analysis (PCA) of descriptors of the candidates, a few substances were selected. The most suitable concentration for each additive was found, and pellets were prepared by an extrusion/spheronisation process. Three pectin grades of different methoxy and amide substitution were tested and the quality of the pellets was evaluated based on size. PLS models were constructed to identify the molecular properties that were most important in producing short, nearly spherical pellets. The results show that quantum chemical descriptors can be a useful tool in the formulation of pectin pellets. Acceptable models relating additive properties and pellet size were achieved. Independent of the pectin grade, the two most important factors favouring formation of small spherical pellets were a small molecular size and a strong hydrogen bond forming ability of the additive molecules.

Sensitive NIRS measurement of increased moisture in stored hygroscopic freeze dried product.

The purpose of this study was to build a best possible NIR prediction model for monitoring of water content in a freeze-dried drug product. The best pre-treatments of the NIR spectra were found to be: transforming from reflection to absorption, baseline correction in the 1845-2165 nm area and a maximum normalisation in the same area. These pre-treatments resulted in a model with the following attributes: SEP of 0.08% (w/w) and one PLS factor, the latter indicating a robust model. The limit of quantification was calculated to 0.24% (w/w). During the stability study an increase in water content in the freeze-dried drug product was revealed, which were found to depend on storage time and temperature. It is believed that the water is derived from the stoppers. The highest increase was found for storage at 40 degrees C, and was estimated to be 0.04% points a month by weight, from an initial value of about 0.25% (w/w).