Aggregation of biopharmaceuticals in human plasma and human serum

Analytical methods based on light microscopy, 90° light-scattering and surface plasmon resonance (SPR) allowed the characterization of aggregation that can occur when antibodies are mixed with human plasma. Light microscopy showed that aggregates formed when human plasma was mixed with 5% dextrose solutions of Herceptin^® (trastuzumab) or Avastin^® (bevacizumab) but not Remicade^® (infliximab). The aggregates in the plasma-Herceptin^®-5% dextrose solution were globular, size range 0.5–9 μm, with a mean diameter of 4 μm. The aggregates in the plasma-Avastin^®-5% dextrose samples had a mean size of 2 μm. No aggregation was observed when 0.9% NaCl solutions of Herceptin^®, Avastin^® and Remicade^® were mixed with human plasma. 90° light-scattering measurements showed that aggregates were still present 2.5 h after mixing Herceptin^® or Avastin^® with 5% dextrose-plasma solution. A SPR method was utilized to qualitatively describe the extent of interactions of surface-bound antibodies with undiluted human serum. Increased binding was observed in the case of Erbitux^® (cetuximab), whereas no binding was measured for Humira^® (adalimumab). The binding of sera components to 13 monoclonal antibodies was measured and correlated with known serum binding properties of the antibodies. The data presented in this paper provide analytical methods to study the intrinsic and buffer-dependent aggregation tendencies of therapeutic proteins when mixed with human plasma and serum.     

Does vacuum-packaging or co-delivered amendments enhance shelf-life of Striga-mycoherbicidal products containing Fusarium oxysporum f. sp. strigae during storage?

The effect of vacuum packaging on the shelf-life and handling of Pesta granules and seed treatment made with chlamydospores of Fusarium oxysporum strains Foxy2, PSM197 or their mixture was studied at 4°C and 22±3°C over 1 year. In addition, the effects of co-incorporated amendments [urea in Pesta or co-delivered fungicides (Ridomil Gold®, Apron XL®) on coated sorghum seeds], and coating material (Arabic gum ‘AG’, SUET Binder ‘SB’) on the viability of Striga-mycoherbicides were evaluated. Storage under vacuum packaging did not enhance shelf-life of the formulated Striga-mycoherbicidal products after 12 months of storage regardless of the treatment used. The co-incorporated urea into Pesta granules significantly reduced the viability of mycoherbicides, but less so at 4°C (58% strain-stability after 12 months). No significant differences between the coating materials in maintaining the viability of mycoherbicides were observed. The shelf-life of isolates on coated seeds significantly decreased when adding Ridomil Gold®. However, at 4°C, the fungicide Apron XL® allowed better survival of Foxy2 and PSM197 by maintaining their averaged half-lives (t _0.5) by an additional 6 months compared to Ridomil Gold®. In general, Striga-mycoherbicidal product combinations exhibited a significantly higher shelf-life when stored at 4°C than at 22±3°C. The absence of a positive effect of vacuum packaging on shelf-life of Striga-mycoherbicidal products reflects the tolerance of the formulated fungal propagules (chlamydospores) to withstand an oxygen enriched environment and allows their handling and distribution through ordinary packaging systems in Africa. The high compatibility between Striga-mycoherbicides and the co-delivered fungicide Apron XL®, and the fungal storage stability allows simultaneous control of Striga and fungal cereal diseases within an integrated pest management (IPM).     

Toxicological implications of a commercial formulation of deltamethrin (Decis®) in developing chick embryo

A deltamethrin containing insecticide formulation (Decis®) was evaluated for its toxic potential in developing chick embryos. For the present study, three water emulsified concentrations of Decis® (12.5 mg L^?1, 25 mg L^?1, and 50 mg L^?1) were used. Fertilized eggs of Gallus domesticus were immersed in these three concentrations of the insecticide for 60 min at 37°C on day 0 of incubation and kept for incubation till embryonic day 7. Recovered embryos were evaluated for teratogenic and biochemical changes. The results revealed that administration of Decis® at its lower concentrations (12.5 mg L^?1 and 25 mg L^?1) did not show any significant teratological changes but the significant number of abnormal survivors was observed at 50 mg L^?1 of dose concentration when compared with vehicle-treated control. Among biochemical changes, total glycogen and RNA contents of embryos was significantly decreased at 25 mg L^?1 and 50 mg L^?1 of Decis® concentrations. Similarly, significant alteration (p ≤ .05) was observed in alanine transaminase activity at 50 mg L^?1 concentration of Decis®. Thus, the present study concluded that the no-effect-level for developmental toxicity for Decis® is below the concentration of 25 mg L^?1 under standard laboratory conditions.     

Micropipette Aspiration‐Based Assessment of Single Channel Water Permeability

Measurements of the unitary hydraulic conductivity of membrane channels, p_f, may be hampered by difficulties in producing sufficient quantities of purified and reconstituted proteins. Low yield expression, the purely empiric choice of detergents, as well as protein aggregation and misfolding during reconstitution may result in an average of less than one reconstituted channel per large unilamellar vesicle. This limits their applicability for p_f measurements, independent of whether light scattering or fluorescence quenching of encapsulated dyes is monitored. Here the micropipette aspiration technique is adopted because its superb sensitivity allows resolving p_f values for one order of magnitude smaller protein densities in sphingomyelin and cholesterol rich giant unilamellar vesicles (GUVs). Protein density is derived from intensity fluctuations that fluorescently labeled channels in the aspirated GUV induce by diffusing through the diffraction limited spot. A perfusion system minimizes unstirred layers in the immediate membrane vicinity as demonstrated by the distribution of both encapsulated and extravesicular aqueous dyes. p_f amounted to 2.4 ± 0.1 × 10^?13 cm³ s^?1 for aquaporin‐1 that served as a test case. The new assay paves the way for directly monitoring the effect that interaction of aquaporins with other proteins or inhibitors may have on p_f on a single sample.     

New therapy with XLQ® to suppress chronic prostatitis through its anti-inflammatory and antioxidative activities

Chronic prostatitis is a common urological disease. The etiology of this disease and effective therapy for its treatment are yet to be elucidated. We investigated the functions of XLQ^® in chronic nonbacterial prostatitis using a complete Freund's adjuvant-induced rat model. Prostates and blood samples were collected for further evaluation after oral gavage with XLQ ^® or a vehicle for 4 weeks. The results showed that XLQ ^® significantly decreased the prostate index, ameliorated the histopathologic changes, and reduced CD3+ and CD45+ cell infiltration in the prostate stroma. Further study showed that XLQ ^® suppressed the expression of proinflammatory cytokines, such as interleukin (IL)-1β, IL-2, IL-6, IL-17A, monocyte chemoattractant protein-1, and tumor necrosis factor-α. XLQ ^® showed a strong antioxidant capacity by enhancing the activities of antioxidative enzymes (e.g., total superoxide dismutase, catalase, and glutathione peroxidase) and decreasing the level of lipid peroxidation products (malondialdehyde). Moreover, XLQ ^® can suppress the activation of nuclear factor-κB and P38-mitogen-activated protein kinase signaling pathways. In summary, XLQ ^® has affirmative effects on chronic prostatitis, which could be attributed to its anti-inflammatory and antioxidative capacities. On the basis of these results, XLQ ^® can be developed as an effective and safe therapy for chronic prostatitis.     

Symmetric transcription of simian virus 40 DNA in the nuclei of transformed mouse cells.

Na⁺ channels from lobster nerve membranes stored frozen in sucrose were incorporated into artificial liposomes. Crude soybean phospholipids or mixtures of purified phospholipids were suitable for reconstitution provided the latter included phosphatidylserine or another acidic phospholipid. The ²²Na flux into the reconstituted vesicles was increased (2 to 3-fold) by veratridine (0.25 – 1 mM) or grayanotoxin I (50 –150 μM) and the increment was abolished by 10 nM tetrodotoxin (K_i = 2 nM). The reconstituted vesicles were inactivated after incubation for 15 min at 40° and exposure to 20 μM dicyclohexylcardobiimide inhibited by 80% the response to the drugs.     

Interaction between succinate dehydrogenase and ubiquinone-binding protein from succinate-ubiquinone reductase

Purified ubiquinone-binding protein in succinate-ubiquinone reductase (QPs) reconstitutes with pure soluble succinate dehydrogenase to form succinate-ubiquinone oxidoreductase upon mixing of the two proteins in phosphate buffer at neutral pH. The maximal reconstitution was found with a weight ratio of succinate dehydrogenase to QPs of about 5, which is fairly close to the calculated value of 6.5, a value obtained by assuming one mole of QPs reacts with one mole of succinate dehydrogenase. Succinate-cytochrome c reductase was reconstituted when succinate dehydrogenase and QPs were added to Complex III or cytochrome b-c₁ III complex (a highly purified ubiquinol-cytochrome c reductase). The reconstituted enzyme possessed kinetic parameters which were identical to those of the native enzyme complex. Interaction between QPs and succinate dehydrogenase resulted in the disappearance of low K_m ferricyanide reductase activity from the latter. Unlike soluble succinate dehydrogenase, the reconstituted enzyme, as well as native succinate-cytochrome c reductase, reduced low concentration ferricyanide only in the presence of excess ubiquinone. The apparent K_m for ubiquinone was 6 μM for reduction of ferricyanide (300 μM) by succinate, which is similar to the K_m when ubiquinone was used as electron acceptor. When 2,6-dichlorophenolindophenol was used as electron acceptor for reconstitution of succinate-ubiquinone reductase very little or no exogeneous ubiquinone was needed to show the maximal activity with QPs made by Method II, indicating that the bound ubiquinone in QPs is enough for enzymatic activity. In addition to restoring the succinate-ubiquinone reductase activity the interaction between QPs and succinate dehydrogenase not only stabilized succinate dehydrogenase but also partially deaggregated QPs. The reconstituted succinate-ubiquinone reductase had a minimal molecular weight of 120000 when the reconstituted system was dispersed in 0.2% Triton X-100. The maximal reconstitution was observed at neutral pH in phosphate buffer, Tris-acetate or Tris-phosphate buffer. Tris-HCl buffer, however, produced a less efficient reconstitution. These results indicate that the interaction between QPs and succinate dehydrogenase may involve some cationic group which has a high affinity for Cl^?. Primary amino groups of QPs are not directly involved in the interaction as the reconstitution showed no significant difference when the amino groups of QPs were alkylated with fluorescamine. The Arrhenius plots of reconstituted succinate-ubiquinone reductase show that the enzyme catalyzes the reaction with an activation energy of 19.7 kcal/mol and 26.6 kcal/mol at temperatures above and below 26°C, respectively. These activation energies are similar to those obtained with native enzyme. The Arrhenius plots of the interaction between QPs and succinate dehydrogenase also have a break point at 26°C. The activation energy for this interaction was calculated to be 11.2 kcal/mol and 6.9 kcal/mol for the temperatures above and below the break-point. The significance of the difference in activation energies between the enzymatic reaction and the reconstitution reaction are further explored in the discussion.     

Evaluation of chromogenic technologies for use in Australian potable water

Aims: To compare the use of MI agar, Membrane Lactose Glucoronide Agar (MLGA), CM1046 agar and Colilert^®-18 (Defined Substrate Technology^®, IDEXX Laboratories Pty. Ltd., Sydney) on Australian potable water. Methods and Results: Both potable (n = 369) and nonpotable waters (n = 35) were analysed by membrane filtration using chromogenic agars as well as Colilert^®-18 over a period of 12 months. Recoveries of stressed organisms on these chromogenic media were also investigated. Agar-based chromogenic technologies compared favourably to Colilert^®-18 for chlorinated waters, but there are possible limitations when using these agars for chloraminated waters. Additionally, the breakthrough of problematic organisms, especially oxidase positive organisms, may lead to misrepresentation or over-estimation of E. coli and total coliforms, particularly on MLGA and CM1046. The recovery of stressed organisms was favoured in the Colilert^®-18 system when compared to chromogenic agars. Conclusions: MI agar performed better than the other chromogenic agars with respect to recovery and colour identification and discrimination of organisms, and compared favourably with Colilert^®-18. The use of chromogenic agars in chloraminated waters should be done cautiously. Significance and Impact of the Study: This study provides comparison data for laboratories looking to adopt chromogenic technologies, and is especially important for Australian laboratories wanting to uptake the use of MI agar (as used in USEPA method 1604) for routine use and for gaining accreditation. Additionally, to the best of our knowledge, this is the first reported evaluation of these agars in chloraminated waters and is especially timely as the use of this disinfection agent is increasing.     

Limiting cell aggregation during mesenchymal stem cell expansion on microcarriers

Mesenchymal stem cells (MSC) are known to be a valuable cell source for tissue engineering and regenerative medicine. However, one of the main limiting steps in their clinical use is the amplification step. MSC expansion on microcarriers has emerged during the last few years, fulfilling the lack of classical T‐flasks expansion. Even if the therapeutic potential of MSC as aggregates has been recently highlighted, cell aggregation during expansion has to be avoided. Thus, MSC culture on microcarriers has still to be improved, notably concerning cell aggregation prevention. The aim of this study was to limit cell aggregation during MSC expansion on Cytodex‐1^®, by evaluating the impact of several culture parameters. First, MSC cultures were performed at different agitation rates (0, 25, and 75 rpm) and different initial cell densities (25 and 50 × 10⁶ cell g^?1 Cytodex‐1^®). Then, the MSC aggregates were put into contact with additional available surfaces (T‐flask, fresh and used Cytodex‐1^®) at different times (before and after cell aggregation). The results showed that cell aggregation was partly induced by agitation and prevented in static cultures. Moreover, cell aggregation was dependent on cell density and correlated with a decrease in the total cell number. It was however shown that the aggregated organization could be dissociated when in contact with additional surfaces such as T‐flasks or fresh Cytodex‐1^® carriers. Finally, cell aggregation could be successfully limited in spinner flask by adding fresh Cytodex‐1^® carriers before its onset. Those results indicated that MSC expansion on agitated Cytodex‐1^® microcarriers could be performed without cell aggregation, avoiding a decrease in total cell number. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Total reconstitution of active small ribosomal subunits of the extreme halophilic archaeon Haloferax mediterranei

The small ribosomal subunit of the halophilic archaeon Haloferax mediterranei has been reconstituted from its dissociated rRNA and protein components. Efficient reconstitution of particles, fully active in poly(U)-dependent polyphenylalanine synthesis, occurs after 2 h of incubation at 36°C in the presence of l.5 M of (NH₄)₂SO₄ 100 mM of MgAc₂, 20 mM Tris-HCI (pH 8.2) and 6 mM 2-mercaptoethanol. Important differences in the optimal ionic conditions for the reconstitution of the 30S and the 50S ribosomal subunits from Haloferax mediterranei have been found. K⁺ and NH₄⁺ ions have differing abilities to promote the reconstitution of the particles. The assembly of 30S ribosomal subunits of H. mediterranei has a higher tolerance to ionic strength than the assembly of the 50S subunits and it is independent of the Mg²⁺concentration present in the system.     

Phage display revisited: Epitope mapping of a monoclonal antibody directed against Neisseria meningitidis adhesin A using the PROFILER technology

There is a strong need for rapid and reliable epitope mapping methods that can keep pace with the isolation of increasingly larger numbers of mAbs. We describe here the identification of a conformational epitope using Phage-based Representation OF ImmunoLigand Epitope Repertoire (PROFILER), a recently developed high-throughput method based on deep sequencing of antigen-specific lambda phage-displayed libraries. A novel bactericidal monoclonal antibody (mAb 9F11) raised against Neisseria meningitidis adhesin A (NadA), an important component of the Bexsero^® anti-meningococcal vaccine, was used to evaluate the technique in comparison with other epitope mapping methods. The PROFILER technology readily identified NadA fragments that were capable of fully recapitulating the reactivity of the entire antigen against mAb 9F11. Further analysis of these fragments using mutagenesis and hydrogen-deuterium exchange mass-spectrometry allowed us to identify the binding site of mAb 9F11 (A250-D274) and an adjoining sequence (V275-H312) that was also required for the full functional reconstitution of the epitope. These data suggest that, by virtue of its ability to detect a great variety of immunoreactive antigen fragments in phage-displayed libraries, the PROFILER technology can rapidly and reliably identify epitope-containing regions and provide, in addition, useful clues for the functional characterization of conformational mAb epitopes.     

Therapy of ankylosing spondylitis and other spondyloarthritides: established medical treatment, anti-TNF-α therapy and other novel approaches

Therapeutic options for patients with more severe forms of spondyloarthritis (SpA) have been rather limited in recent decades. There is accumulating evidence that anti-tumor-necrosis-factor (anti-TNF) therapy is highly effective in SpA, especially in ankylosing spondylitis and psoriatic arthritis. The major anti-TNF-α agents currently available, infliximab (Remicade^®) and etanercept (Enbrel^®), are approved for the treatment of rheumatoid arthritis (RA) in many countries. In ankylosing spondylitis there is an unmet medical need, since there are almost no disease-modifying antirheumatic drugs (DMARDs) available for severely affected patients, especially those with spinal manifestations. Judging from recent data from more than 300 patients with SpA, anti-TNF therapy seems to be even more effective in SpA than in rheumatoid arthritis. However, it remains to be shown whether patients benefit from long-term treatment, whether radiological progression and ankylosis can be stopped and whether long-term biologic therapy is safe.     

Reconstitution of Pronase-Susceptible Flocs of Flavobacterium sp.

A component responsible for the aggregation of cells was extracted from Flavobacterium strain B by treatment of cells with 5 m guanidine hydrochloride and partially purified by gel filtration. The guanidine hydrochloride-extracted cells were reaggregated with the component after dialysis against 0.3mm of CaCl₂. Various divalent cations were effective in place of Ca²⁺, but Ca²⁺ was most effective for reconstitution. The reconstituted flocs were deflocculated by the treatment of Pronase or ethylenediaminetetraacetic acid indicating that reconstituted flocs closely resemble natural flocs.     

A rapid and simple chemiluminescence method for screening levels of inosine and hypoxanthine in non‐traumatic chest pain patients

A rapid and simple chemiluminescence method was developed for detection of inosine and hypoxanthine in human plasma. The method utilized a microplate luminometer with direct injectors to automatically dispense reagents during sample analysis. Enzymatic conversions of inosine to hypoxanthine, followed by hypoxanthine to xanthine to uric acid, generated superoxide anion radicals as a useful metabolic by‐product. The free radicals react with Pholasin^®, a sensitive photoprotein used for chemiluminescence detection, to produce measurable blue‐green light. The use of Pholasin^® and a chemiluminescence signal enhancer, Adjuvant‐K?, eliminated the need for plasma clean‐up steps prior to analysis. The method used 20 μL of heparinized plasma, with complete analysis of total hypoxanthine levels (inosine is metabolized to hypoxanthine using purine nucleoside phosphorylase) in approximately 3.7 min. The rapid chemiluminescence method demonstrated the capability of differentiating total hypoxanthine levels between healthy individuals, and patients presenting with non‐traumatic chest pain and potential acute cardiac ischemia. The results support the potential use of chemiluminescence methodology as a diagnostic tool to rapidly screen for elevated levels of inosine and hypoxanthine in human plasma, potential biomarkers of acute cardiac ischemia.Copyright ©2009 John Wiley & Sons, Ltd.     

Lyophilization of TC-99m-Hynic Labeled Peg-Liposomes

Abstract

The development of long circulating liposomes represented a major step forward towards the use of radiolabeled liposomes in nuclear medicine. The long circulation property markedly improves their uptake and consequently visualization of sites of infection and inflammation. Previously, we have developed a rapid and convenient method to label polyethylene glycol (PEG)-lipo-somes with technetium-99m (Tc-99m). PEG-liposomes containing the technetium-chelator hydrazino nicotinamide (HYNIC) could be labeled with Tc-99m with high efficiency. We showed that these Tc-99m-HYNIC labeled PEG-liposomes have excellent in vivo imaging characteristics in several pre-clinical and clinical studies. However, an important limitation associated with the use of HYNIC-PEG-liposome formulation as radiopharmaceutical is that their labeling efficiency decreases markedly within 3 months. In this paper we present a lyophilization method for HYNIC-PEG-liposomes using sucrose as a lyopro-tectant. The long-term stability of these liposomes in terms of the particle size and labeling efficiency upon reconstitution were determined. Additionally, the in vivo behavior of reconstituted radiolabeled liposomes in a rat model of focal infection was studied at two time-points after preparation.

Increasing the duration of the dehydration step significantly reduced the mean particle size upon reconstitution. Increasing the storage temperature from -20^°C to +4^°C also improved the particle size distribution upon reconstitution. The labeling efficiency for both freeze-dried preparations remained high during the 1 year-storage period and was always higher than 86%, but decreased for the control liposomes. Eight months after preparation, these liposomes had a labeling efficiency as low as 6%, whereas both freeze-dried preparations could still be labeled with an efficiency of 90%. The in vivo studies showed that there was no major difference in the biodistribution of the radiolabeled liposomes between 3 and 30 weeks post-preparation in rats with an Staphylococcus aureus abscess, indicating an acceptable long-term shelf-life of both freeze-dried liposome preparations. Abscesses were visualized from 2 hours post injection onwards.

In conclusion, a freeze-drying method which improved the long term shelf-life of HYNIC-PEG-liposomes is presented. The in vivo behavior of Tc-99m-PEG-liposomes, reconstituted 30 weeks after preparation, was similar to the biodistribution obtained with the non-freeze-dried preparation. The splenic uptake of these liposomes was slightly increased.     

Mechanism of the action of biogenic chloramines and hypochlorite on the initial aggregation of platelets

The antiaggregant effect of two reactive oxidants—N,N-dichlorotaurine (a biogenic chloramine) and sodium hypochlorite—on the initial ADP-induced aggregation of rabbit blood platelets was studied. Platelet aggregation in reconstituted platelet-rich plasma was measured nephelometrically; an increase in the intensity of small-angle light scattering served as an index of aggregation. Addition of chloramine at relatively small concentrations (no greater than 1 mM available chlorine) directly to the reconstituted platelet-rich plasma suppressed the initial aggregation (formation of small aggregates) several times more strongly than preincubation of native plasma with chloramine. This suggests that N,N-dichlorotaurine realizes its antiaggregant effect on the platelet-rich plasma by directly interacting with cells. The effects of the inhibition of platelet aggregation in two variants of addition of high concentrations of N,N-dichlorotaurine did not differ significantly. In this case, a large amount of residual unreacted chloramine remained in the plasma, which caused the suppression of platelet aggregation during subsequent reconstitution of the platelet-rich plasma. Similar data were obtained in studying the antiaggregant effect of hypochlorite. N,N-Dichlorotaurine and hypochlorite at concentrations of 0.2–0.3 and 0.15 mM, respectively, strongly inhibited the initial aggregation of isolated platelets (approximately 2·10⁸ cells/ml) preliminarily activated for 1.5 min by addition of 0.1–0.5 μM ADP. However, the antiaggregants had a more profound suppressive effect on the aggregation of unstimulated platelets. The antiaggregant effects of N,N-dichlorotaurine and hypochlorite probably stem from the oxidative modification of the sulfur-containing groups in platelet plasma membrane.     

“Aqua-space®”, a New Headspace Method for Isolation of Natural Floral Aromas Using Humidified Air as a Carrier Gas

A new method called “Aqua-space^®” was developed for the isolation of the natural fragrances of plants. Living flowers were enclosed in a space under simulated natural conditions, and humidified air was pumped into the space as a fragrance carrier. In a comparison among three isolation methods, i.e., Aqua-space^®, headspace, and solvent extraction, the Aqua-space^® method proved to be the most effective in retaining natural fragrances with abundant oxygenated components key to floral fragrances.     

A study of the concept of non-radioactive unit-dosed reagent kits [cold unit doses (CUDs)] as an efficient and cost-saving method for 99mTc radiopharmaceutical preparation

Traditionally, when preparing ^99mTc-labeled radiopharmaceuticals, [^99mTc]pertechnetate is added to the entire contents of a vial of reagent kit, and patient doses are subsequently withdrawn from the vial. This technique of compounding can be potentially wasteful for two reasons: (1) once reconstituted with ^99mTc, most reagent kits have a relatively short shelf-life, and thus the entire contents may not be used before expiration and (2) due to a need to conserve radioactivity in many hospitals, enough [^99mTc]pertechnetate is added to the reagent kit in order to retrieve only 1–2 patient doses, even though adequate chemicals (ligand, reducing agent, etc.) are present in the reagent kit to supply as many as 5–10 doses. Hence, a method for optimizing the efficient use of reagent kits would be desirable. The purpose of this study was to determine the feasibility of unit-dosing non-radioactive reagent kits and storing these cold unit doses (CUDs) for eventual labeling with ^99mTc. To evaluate this concept, unit doses were prepared from reagent kits of medronate (MDP) and pentetate (DTPA). The specific variables studied in this research were the effects of storage time, storage temperature and reconstitution volume (dilution) on the unit doses. These effects were monitored by measuring the radiochemical and biodistribution properties of the unit doses following their final reconstitution with [^99mTc]pertechnetate. The labeling efficiency was determined using instant thin layer chromatography (ITLC), and the biodistribution patterns of these radiolabeled CUDs were studied in mice. The results showed that MDP- and DTPA-CUDs stored at −18 °C retained the properties which resulted in acceptable radiochemical purity and biodistribution in mice for as long as 30 days. On the other hand, the radiochemical purity of MDP and DTPA unit doses stored at 25 °C deteriorated rapidly. Mean radiochemical purities as low as 0.58–19.4% were observed on day 30. Altered biodistributions were observed in a manner consistent with the decreased labeling efficiencies. The CUDs of lower dilution (3 mL) appeared to be more stable than the CUDs of higher dilution (10 mL). However, the effect of reconstitution volume was much less significant than the temperature effect on the CUDs. In conclusion, the concept of unit-dosing non-radioactive reagent kits appears to provide an efficient and cost-saving method for preparing infrequent and emergency radiopharmaceutical doses. The study also showed that the storage temperature of these unit doses is critical to the success of the procedure. The volume of reconstitution has a minimal impact on the stability of CUDs if stored at the appropriate temperature.     

Evaluation of the in vitro differential protein adsorption patterns of didanosine-loaded nanostructured lipid carriers (NLCs) for potential targeting to the brain

The preferential in vitro adsorption of apolipoprotein E (Apo E) onto the surface of colloidal drug carriers may be used as a strategy to evaluate the in vivo potential for such systems to transport drugs to the brain. The aim of this research was to investigate the in vitro protein adsorption patterns of didanosine-loaded nanostructured lipid carriers (DDI-NLCs), using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), in order to establish the potential for NLCs to deliver DDI to the brain. NLC formulations were manufactured using high-pressure homogenization using a lipid matrix consisting of a mixture of Precirol^® ATO 5 and Transcutol^® HP. The 2-D PAGE analysis revealed that NLCs in formulations stabilized using Solutol^® HS 15 alone or with a ternary surfactant system consisting of Solutol^® HS 15, Tween^® 80, and Lutrol^® F68, preferentially adsorbed proteins, such as Apo E. Particles stabilized with Tween^® 80 and Lutrol^® F68 did not adsorb Apo E in these studies, which could be related to the relatively large particle size and hence small surface area observed for these NLCs. These findings have revealed that DDI-loaded NLCs may have the potential to deliver DDI to the brain in vivo and, in addition, to Tween^® 80, which has already been shown to have the ability to facilitate the targeting of colloidal drug delivery systems to the brain. Solutol^® HS 15–stabilized nanoparticles may also achieve a similar purpose.     

A comparison of biophysical characterization techniques in predicting monoclonal antibody stability

With the rapid growth of biopharmaceutical product development, knowledge of therapeutic protein stability has become increasingly important. We evaluated assays that measure solution-mediated interactions and key molecular characteristics of 9 formulated monoclonal antibody (mAb) therapeutics, to predict their stability behavior. Colloidal interactions, self-association propensity and conformational stability were measured using effective surface charge via zeta potential, diffusion interaction parameter (k_D) and differential scanning calorimetry (DSC), respectively. The molecular features of all 9 mAbs were compared to their stability at accelerated (25°C and 40°C) and long-term storage conditions (2–8°C) as measured by size exclusion chromatography. At accelerated storage conditions, the majority of the mAbs in this study degraded via fragmentation rather than aggregation. Our results show that colloidal stability, self-association propensity and conformational characteristics (exposed tryptophan) provide reasonable prediction of accelerated stability, with limited predictive value at 2–8°C stability. While no correlations to stability behavior were observed with onset-of-melting temperatures or domain unfolding temperatures, by DSC, melting of the Fab domain with the C_H2 domain suggests lower stability at stressed conditions. The relevance of identifying appropriate biophysical assays based on the primary degradation pathways is discussed.