A Critical Review of Biological Properties, Delivery Systems and Analytical/Bioanalytical Methods for Determination of Bevacizumab.

Bevacizumab is a chimeric monoclonal human-murine antibody originated from murine monoclonal antibody (muMAb A4.6.1) with the human immunoglobulin IgG1. BVZ binds the extracellular portion of vascular endothelial growth factor receptors (VEGFR), which have tyrosine kinase activity. The mechanism of action of BVZ involves binding to VEGFR, Flt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2), inducing homodimerization of two receptor subunits, and, consequently, autophosphorylation of their tyrosine kinase domains located inside the cytoplasm. With the advent of nanostructured systems it is increasingly necessary to look for safe analytical methods, ensuring the reliability of the results obtained by them, becoming essential to ensure the quality of medicines. In this work, the incorporation of bevacizumab in to different drug delivery systems was presented. Moreover, detailed investigation was performed about methods for qualitative and quantitative analyses of bevacizumab, including, biological fluids, and drug delivery systems, were investigated. Most recently high performance liquid chromatography coupled with various detectors, liquid chromatography, mass spectrometry and ELISA were used for this purpose. Thus, this review was performed to evaluate the benefits of bevacizumab carried by nanostructured systems and the analytical methods available for detection and quantification of these drugs.

In-depth analysis of monoclonal antibodies using microfluidic capillary electrophoresis and native mass spectrometry.

Charge variant profiling of therapeutic proteins is required by the International Council for Harmonisation guidelines and is traditionally performed by capillary electrophoresis or ion exchange chromatography. Recently, improvements in the hyphenation of capillary electrophoresis with mass spectrometry and the introduction of mass spectrometry compatible background electrolytes has allowed the implementation of native mass spectrometric determination of the charge variant profile obtained from the electrophoretic separation. The low flow operation of the microfluidic electrophoretic platform significantly boosts mass spectrometric sensitivity and increases the dynamic range, even when using sample amounts as low as 1 ng in capillary. In the current study, rituximab, trastuzumab and bevacizumab drug products were analysed using the ZipChip microfluidic CE-ESI-MS platform that facilitated confident identification of proteoforms with an average mass accuracy of <15 ppm. Up to 52 proteoforms were identified for trastuzumab drug product, while rituximab sample revealed the presence of fragments and sialylated N-glycans. Overall, the CE-ESI-MS platform proved to be a fast and robust tool for therapeutic protein charge variant profiling and facilitated efficient coupling with native mass spectrometry for the generation of highly informative characterisation data.

[Authentication study of the preparation 'Avastin' by means of chemical-toxicological analysis]. / Issledovanie podlinnosti preparata 'Avastin' metodami khimiko-toksikologicheskogo analiza.

Falsification and use of low-quality drugs of biological origin creates a threat to public health. To a greater extent, costly drugs, including bevacizumab, are exposed to similar abuses. Timely determination of cases of forgery or the improper clinical use of monoclonal antibody preparations is one of the necessary measures that can be taken to limit the risks and preserve the health of patients. This paper presents the results of the investigation of the bevacizumab preparation 'Avastin', which was withdrawn from ophthalmic clinical practice in the course of the investigation. We compared the qualitative and quantitative composition of the drug samples, which were determined using commonly available methods of chemical and toxicological analysis.

A validated size exclusion chromatography method coupled with fluorescence detection for rapid quantification of bevacizumab in ophthalmic formulations.

Bevacizumab is a full-length human monoclonal antibody used to treat various neovascular diseases such as wet age-related macular degeneration (AMD), diabetic eye disease and other problems of the retina. Monthly intravitreal injections of bevacizumab (Avastin®) are effective in the treatment of wet AMD. However, there is a growing demand in the development of sustained release ophthalmic formulations. Therefore, this study aims, for the first time, to develop a rapid, simple, and sensitive method using size exclusion chromatography coupled with fluorescence detection for routine quantification of bevacizumab in ophthalmic formulations and during in vitro release studies. The selected chromatographic conditions included an aqueous mobile phase composed of 35 mM sodium phosphate buffer and 300 mM sodium chloride (pH 6.8), a flow rate of 0.5 mL/min, and the fluorescence detector was operated at excitation and emission wavelengths of 280 and 340 nm, respectively. The peak area-concentration relationship maintained its linearity over concentration range of 0.1-20 µg/mL (R2 = 0.9993), and the quantitation limit was 100 ng/mL. The method was validated for specificity, accuracy, precision, and robustness. The developed method had a run time of 6 min at temperature 25 °C, making it a unique validated method for rapid and cost-effective quantification of bevacizumab.

Validation of Rapi-Fluor method for glycan profiling and application to commercial antibody drugs.

N-glycans influence the activity of antibody drugs such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Thus, glycan profiling is considered a critical quality attribute (CQA) and requires routine and comprehensive monitoring. In this report, we validate the new glycan profiling method called Rapi-Fluor method, which reduced the sample preparation time and increased the FLR and MS intensities compared with conventional 2-AB method. Optimized glycan release, labeling, hydrophilic interaction liquid chromatography (HILIC) enrichment, and HILIC separation resulted in low variation and short preparation time. The method evaluated for human IgG standard varied from 100 µg/mL to 4000 µg/mL in 25 µL of water. The determination of coefficient (r2 > 0.9992), recovery (88.992% ~ 111.198%), limit of detection (LOD < 193.274 µg/mL), limit of quantification (LOQ < 585.679 µg/mL), and precision (Intra-day < 2.317%RSD and Inter-day < 4.287%RSD) were evaluated with four major glycans from antibody drugs. In addition, the method was used for glycan profiling of five different commercial antibodies. The method yielded precise results for IgG glycan analysis and demonstrated effective glycan profiling of commercial antibody drugs.

A Novel Online Four-Dimensional SEC×SEC-IM×MS Methodology for Characterization of Monoclonal Antibody Size Variants.

The determination of size variants is a major critical quality attribute of a therapeutic monoclonal antibody (mAb that may affect the drug product safety, potency, and efficacy. Size variant characterization often relies on size-exclusion chromatography (SEC), which could be hampered by difficult identification of peaks. On the other hand, mass spectrometry (MS)-based techniques performed in nondenaturing conditions have proven to be valuable for mAb-related compound characterization. On the basis of the observation that limited SEC performance was observed in nondenaturing MS compatible ammonium acetate buffer compared with classical phosphate salts, a multidimensional analytical approach was proposed. It combines comprehensive online two-dimensional chromatography (SEC×SEC), with ion mobility and mass spectrometry (IM-MS) in nondenaturing conditions for the characterization of a variety of mAbs. We first exemplify the versatility of our approach for simultaneous detection, identification, and quantitation of adalimumab size variants. Benefits of the SEC×SEC-native IM×MS were further highlighted on forced degraded pembrolizumab and bevacizumab samples, for which the 4D setup was mandatory to obtain an extensive and unambiguous identification, and accurate quantitation of unexpected high/low molecular weight species (HMWS and LMWS). In this specific context, monomeric conformers were detected by IM-MS as HMWS or LMWS. Altogether, our results emphasize the power of comprehensive 2D LC×LC setups hyphenated to IM×MS in nondenaturing conditions with unprecedented performance including: (i) maintaining optimal SEC performance (under classical nonvolatile salt conditions), (ii) performing online native MS identification, and (iii) providing IM-MS conformational characterization of all separated size variants.

Development and validation of generic heterogeneous fluoroimmunoassay for bioanalysis of bevacizumab and cetuximab monoclonal antibodies used for cancer immunotherapy.

This study describes, for the first time, the development and validation of a highly selective and sensitive heterogeneous fluoroimmunoassay (FIA) for the bioanalysis of two monoclonal antibodies (mAbs) used for cancer immunotherapy: bevacizumab (BEV) and cetuximab (CET). The assay combines reliable non-competitive binding of BEV and CET to their specific cell receptor proteins (human vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR), respectively) with the highly specific fluorescence activity of the fluorescein isothiocyanate labeled anti-human IgG (FITC-IgG) used as label. The limits of detection were 14.14 and 1.27 × 103 ng mL-1 for BEV and CET, respectively. The accuracy and precision of the assay were demonstrated. The assay is simple, convenient, and requires very small volume (~ 5 µL) of plasma sample for analysis. The assay can offer high throughput analysis in clinical settings when modern microplates of multiplies of 96 (up to 6144-wells) are used and/or integrated as a part of automated robotic system. The proposed assay can be used for routine clinical bioanalysis of mAbs with potential application in pharmacokinetics, pharmacodynamics and therapeutic drug monitoring (TDM).

Costs and effects of intra-operative fluorescence molecular imaging - A model-based, early assessment.

INTRODUCTION: Successful breast conserving cancer surgeries come along with tumor free resection margins and account for cosmetic outcome. Positive margins increase the likelihood of tumor recurrence. Intra-operative fluorescence molecular imaging (IFMI) aims to focus surgery on malignant tissue thus substantially lowering the presence of positive margins as compared with standard techniques of breast conservation (ST). A goal of this paper is to assess the incremental number of surgeries and costs of IFMI vs. ST. METHODS: We developed a decision analytical model and applied it for an early evaluation approach. Given uncertainty we considered that IFMI might reduce the proportion of positive margins found by ST from all to none and this proportion is assumed to be reduced to 10% for the base case. Inputs included data from the literature and a range of effect estimates. For the costs of IFMI, respective cost components were added to those of ST. RESULTS: The base case reduction lowered number of surgeries (mean [95% confidence interval]) by 0.22 [0.15; 0.30] and changed costs (mean [95% confidence interval]) by €-663 [€-1,584; €50]. A tornado diagram identified the Diagnosis Related Group (DRG) costs, the proportion of positive margins of ST, the staff time saving factor and the duration of frozen section analysis (FSA) as important determinants of this cost. CONCLUSIONS: These early results indicate that IFMI may be more effective than ST and through the reduction of positive margins it is possible to save follow-up surgeries-indicating further health risk-and to save costs through this margin reduction and the avoidance of FSA.

Simple and ultra-fast recognition and quantitation of compounded monoclonal antibodies: Application to flow injection analysis combined to UV spectroscopy and matching method.

Compounding of monoclonal antibody (mAbs) constantly increases in hospital. Quality control (QC) of the compounded mAbs based on quantification and identification is required to prevent potential errors and fast method is needed to manage outpatient chemotherapy administration. A simple and ultra-fast (less than 30 s) method using flow injection analysis associated to least square matching method issued from the analyzer software was performed and evaluated for the routine hospital QC of three compounded mAbs: bevacizumab, infliximab and rituximab. The method was evaluated through qualitative and quantitative parameters. Preliminary analysis of the UV absorption and second derivative spectra of the mAbs allowed us to adapt analytical conditions according to the therapeutic range of the mAbs. In terms of quantitative QC, linearity, accuracy and precision were assessed as specified in ICH guidelines. Very satisfactory recovery was achieved and the RSD (%) of the intermediate precision were less than 1.1%. Qualitative analytical parameters were also evaluated in terms of specificity, sensitivity and global precision through a matrix of confusion. Results showed to be concentration and mAbs dependant and excellent (100%) specificity and sensitivity were reached within specific concentration range. Finally, routine application on "real life" samples (n = 209) from different batch of the three mAbs complied with the specifications of the quality control i.e. excellent identification (100%) and ±â€¯15% of targeting concentration belonging to the calibration range. The successful use of the combination of second derivative spectroscopy and partial least square matching method demonstrated the interest of FIA for the ultra-fast QC of mAbs after compounding using matching method.

Validated reverse phase HPLC diode array method for the quantification of intact bevacizumab, infliximab and trastuzumab for long-term stability study.

The aim of the present study was to develop suitable and reliable method for quantification three of the most worldwide used therapeutic monoclonal antibodies (mAbs) -bevazizumab (BVZ), infliximab (INF) and trastuzumab (TTZ)- to be used in long-term stability studies. Reverse phase (RP) was selected by its greater sensibility and reproducibility comparing with other chromatographic modes. Then a high performance liquid chromatography with diode array detection (RP)HPLC/DAD method was checked. Since the three mAbs represent the active ingredient in the medicines in which they are formulated, the selected method was validated for each one in accordance with the International Conference on Harmonization (ICH) guidelines for pharmaceuticals for human use. Then method was validated in terms of linearity, accuracy, precision, (repeatability, intermediate precision) specificity (by forced degradation studies), robustness and system suitability. Spectral peak purity analysis strategy was used to test mAb degradations. Comparative study of the results indicated similar behavior for the three mAbs. Forced degradation studies also provided deep knowledge of these important bio-macromolecules. At last the method was successfully used to quantify BVZ, INF and TTZ in long-term stability studies performed under hospital conditions of use and they showed great stability regarding quantification during the time of the study.

A Proximity Extension Assay (PEA)-based method for quantification of bevacizumab.

INTRODUCTION: Proximity Extension Assay (PEA) is a direct one-step protein quantification method using a pair of DNA oligonucleotides linked to antibodies against the target molecule. It requires polyclonal or two monoclonal antibodies (mAbs) that bind to target epitopes close enough to form a DNA duplex which is quantified by real-time PCR. Bevacizumab, an anti-cancer drug, is a mAb against vascular endothelial growth factor with common cardiovascular adverse effects. It is widely used off-label to treat neovascular eye disorders by intravitreal application of small doses. Even then, certain amount reaches systemic circulation which is considered relevant regarding safety. We aimed to set-up a PEA-based assay for bevacizumab in human plasma and to preliminary evaluate it in patients treated intravitreally. METHODS: We tested (PEA, quantitative PCR) several combinations of commercial mAbs and a Fab fragment against bevacizumab. The best combination was used to quantify bevacizumab in three patients donating plasma before and 24 h after the first intravitreal injection. RESULTS: A combination of a mAb and a Fab fragment (HCA184 and HCA182, Bio-Rad Laboratories, Inc.) performed best: standard curve R2 0.98, linear dynamic range 1-1000 pM, lower limit of quantification 1 pM (149 pg/mL) and a satisfactory precision (coefficient of variation 12%). All pre-dose patient concentrations were zero, while post-dose concentrations were 10.94, 13.73 and 55.49 ng/mL, in line with previous reports. DISCUSSION: This is the first set-up of a PEA-based assay for quantification of bevacizumab in human plasma. Its good performance and high sensitivity support further evaluation for potential uses particularly when the expected concentrations are low.

Quality of bevacizumab (Avastin®) repacked in single-use glass vials for intravitreal administration.

PURPOSE:: Avastin® (bevacizumab) is an anti-vascular endothelial growth factor (VEGF) monoclonal antibody given as an off-label drug by intravitreal administration for treatment of ocular diseases. The drug's clinical application and its cost-benefit profile has generated demand for its division into single-use vials to meet the low volume and low-cost doses necessary for intraocular administration. However, the safety of compounding the drug in single-use vials is still under discussion. In this study, the stability and efficacy of Avastin® repacked in individual single-use glass vials and glass ampoules by external compounding pharmacies were evaluated. METHODS:: Polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC), dynamic light scattering (DLS), and turbidimetry were selected to detect the formation of aggregates of various sizes. Changes in bevacizumab biological efficacy were investigated by using an enzyme-linked immunosorbent assay (ELISA). RESULTS:: Repacked and reference bevacizumab showed similar results when analyzed by PAGE. By SEC, a slight increase in high molecular weight aggregates and a reduction in bevacizumab monomers were observed in the products of the three compounding pharmacies relative to those in the reference bevacizumab. A comparison of repacked and reference SEC chromatograms showed that the mean monomer loss was ≤1% for all compounding pharmacies. Protein aggregates in the nanometer- and micrometer-size ranges were not detected by DLS and turbidimetry. In the efficacy assay, the biological function of repacked bevacizumab was preserved, with <3% loss of VEGF binding capacity relative to that of the reference. CONCLUSION:: The results showed that bevacizumab remained stable after compounding in ampoules and single-use glass vials; no significant aggregation, fragmentation, or loss of biological activity was observed.

Quality of bevacizumab (Avastin®) repacked in single-use glass vials for intravitreal administration / Avaliação da qualidade do bevacizumabe (Avastin®) fracionado em frascos de vidro de dose única para administração intravítrea

ABSTRACT Purpose: Avastin® (bevacizumab) is an anti-vascular endothelial growth factor (VEGF) monoclonal antibody given as an off-label drug by intravitreal administration for treatment of ocular diseases. The drug's clinical application and its cost-benefit profile has generated demand for its division into single-use vials to meet the low volume and low-cost doses necessary for intraocular administration. However, the safety of compounding the drug in single-use vials is still under discussion. In this study, the stability and efficacy of Avastin® repacked in individual single-use glass vials and glass ampoules by external compounding pharmacies were evaluated. Methods: Polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC), dynamic light scattering (DLS), and turbidimetry were selected to detect the formation of aggregates of various sizes. Changes in bevacizumab biological efficacy were investigated by using an enzyme-linked immunosorbent assay (ELISA). Results: Repacked and reference bevacizumab showed similar results when analyzed by PAGE. By SEC, a slight increase in high molecular weight aggregates and a reduction in bevacizumab monomers were observed in the products of the three compounding pharmacies relative to those in the reference bevacizumab. A comparison of repacked and reference SEC chromatograms showed that the mean monomer loss was ≤1% for all compounding pharmacies. Protein aggregates in the nanometer- and micrometer-size ranges were not detected by DLS and turbidimetry. In the efficacy assay, the biological function of repacked bevacizumab was preserved, with <3% loss of VEGF binding capacity relative to that of the reference. Conclusion: The results showed that bevacizumab remained stable after compounding in ampoules and single-use glass vials; no significant aggregation, fragmentation, or loss of biological activity was observed.

RESUMO Objetivos: Avastin® (bevacizumabe) é um anticorpo monoclonal inibidor do fator de crescimento endotelial de vasos (VEGF) utilizado "off-label" por meio de administração intravítrea para o tratamento de doenças oculares. A sua aplicação clínica associada ao custo-benefício do medicamento gerou uma demanda para seu fracionamento em frascos de dose única para utilização pela via intraocular. No entanto, a segurança do fracionamento do anticorpo em frascos de dose única ainda é alvo de discussão. Neste trabalho, a estabilidade e a eficácia do Avastin® fracionado em frascos ou ampolas de vidro de dose unitária por farmácias de manipulação do mercado foram avaliadas. Métodos: As técnicas de eletroforese em gel de poliacrilamida (PAGE), cromatografia por exclusão de tamanho (SEC), espalhamento dinâmico da luz (DLS) e turbidimetria foram empregadas para avaliar a formação de agregados de diferentes tamanhos. Alterações na atividade biológica do bevacizumabe foram estudadas utilizando ELISA. Resultados: Amostras referência e do bevacizumabe fracionado apresentaram resultados semelhantes quando analisado por gel de poliacrilamida. Por cromatografia por exclusão de tamanho, um pequeno aumento na quantidade de agregados de alta massa molar seguido de uma redução nos monômeros do bevacizumabe foram observados para as amostras das três farmácias de manipulação quando comparado ao referência. A comparação dos cromatogramas mostrou uma quantidade de redução do monômero inferior a 1% para todas as amostras fracionadas. Por espalhamento dinâmico da luz e turbidimetria, não foram detectados agregados de proteína na faixa de tamanho de micrômetro e nanômetro. No ensaio de eficácia, o bevacizumabe fracionado preservou sua função biológica pois apresentou menos de 3% de perda na capacidade de ligação ao VEGF quando comparado ao referência. Conclusão: Este estudo sugere que o bevacizumabe se mantem estável após fracionamento em ampolas e frascos de vidro de dose unitária pois não foram observadas agregação e/ou fragmentação de proteínas e perda de atividade biológica em quan tidades significativas.

Analysis of therapeutic monoclonal antibody glycoforms by mass spectrometry for pharmacokinetics study.

Monoclonal antibodies (mAbs), are one of the most important protein drugs have attracted increasing attention. However, the pharmacokinetics of mAbs has not been fully investigated due to the complexity of protein drugs. Traditonal immuno-based approaches can not recognize the proteoforms of mAbs because of the long development cycles, prohibitive cost, and interactions between different proteins. Therefore, reliable qualitative and quantitative analysis of the proteoforms of mAbs in biological samples is of crucial importance. Herein, a novel method was developed for absolute quantitation of mAbs and their glycoforms in complex biological samples such as serum and tissues. With the combination of HILIC enrichment and parallel reaction monitoring by high resolution mass spectrometry, most of the glycoforms can be accurately quantified at the fmol level through the use of the model mAb of bevacizumab. More importantly, the structural confirmation can be achieved simultaneously without the need for additional experiments. This strategy can be readily applied to the pharmacokinetic study of glycosylation modification and biomarker discovery for clinical applications.

Postmortem vitreous bevacizumab levels of an infant treated for retinopathy of prematurity.

We report the vitreous concentration of bevacizumab after injection for the treatment of retinopathy of prematurity (ROP). A premature neonate diagnosed with type 1 ROP was treated in both eyes with 0.625 mg intravitreal bevacizumab injection at 32 weeks' postconceptual age. Eleven weeks later there was complete regression clinically, but the patient died. Vitreous samples taken at autopsy revealed a bevacizumab vitreous concentration of 41.57 ng/ml. Histopathology of the retina showed residual preretinal neovascularization. Bevacizumab elimination from the infant vitreous is similar to that of adults, and, although complete regression was clinically apparent, it was not confirmed histopathologically.

High-throughput identification of monoclonal antibodies after compounding by UV spectroscopy coupled to chemometrics analysis.

Monoclonal antibodies (mAbs) compounded into the hospital pharmacy are widely used nowadays. Their fast identification after compounding and just before administration to the patient is of paramount importance for quality control at the hospital. This remains challenging due to the high similarity of the structure between mAbs. Analysis of the ultraviolet spectral data of four monoclonal antibodies (cetuximab, rituximab, bevacizumab, and trastuzumab) using unsupervised principal component analysis led us to focus exclusively on the second-derivative spectra. Partial least squares-discriminant analysis (PLS-DA) applied to these data allowed us to build models for predicting which monoclonal antibody was present in a given infusion bag. The calibration of the models was obtained from a k-fold validation. A prediction set from another batch was used to demonstrate the ability of the models to predict well. PLS-DA models performed on the spectra of the region of aromatic amino acid residues presented high ability to predict mAb identity. The region corresponding to the tyrosine residue reached the highest score of good classification with 89 %. To improve the score, standard normal variate (SNV) preprocessing was applied to the spectral data. The quality of the optimized PLS-DA models was enhanced and the region from the tyrosine/tryptophan residues allowed us excellent classification (100 %) of the four mAbs according to the matrix of confusion. The sensitivity and specificity performance parameters assessed this excellent classification. The usefulness of the combination of UV second-derivative spectroscopy to multivariate analysis with SNV preprocessing demonstrated the unambiguous identification of commercially available monoclonal antibodies. Graphical abstract PLS-DA models on the spectra of the region of aromatic amino acid residues allows mAb identification with high prediction.

Hydrodynamic Radii of Ranibizumab, Aflibercept and Bevacizumab Measured by Time-Resolved Phosphorescence Anisotropy.

PURPOSE: To measure the hydrodynamic radii of intravitreal anti-VEGF drugs ranibizumab, aflibercept and bevacizumab with µs time-resolved phosphorescence anisotropy. METHODS: Ruthenium-based dye Ru(bpy)2(mcbpy - O - Su - ester)(PF6)2, whose lifetime of several hundred nanoseconds is comparable to the rotational correlation time of these drugs in buffer, was used as a label. The hydrodynamic radii were calculated from the rotational correlation times of the Ru(bpy)2(mcbpy - O - Su - ester)(PF6)2-labelled drugs obtained with time-resolved phosphorescence anisotropy measurements in buffer/glycerol solutions of varying viscosity. RESULTS: The measured radii of 2.76±0.04 nm for ranibizumab, 3.70±0.03 nm for aflibercept and 4.58±0.01 nm for bevacizumab agree with calculations based on molecular weight and other experimental measurements. CONCLUSIONS: Time-resolved phosphorescence anisotropy is a relatively simple and straightforward method that allows experimental measurement of the hydrodynamic radius of individual proteins, and is superior to theoretical calculations which cannot give the required accuracy for a particular protein.

Pre-study and in-study validation of a size-exclusion chromatography method with different detection modes for the analysis of monoclonal antibody aggregates.

Size exclusion chromatography (SEC) with different detection modes was assessed as a means to characterize the type of bevacizumab aggregate that forms under thermal stress, quantitatively monitoring the aggregation kinetics. The combination of SEC with light-scattering (SEC/LS) detection was validated using in-study validation process. This was performed by applying a strategy based on a control chart to monitor the process parameters and by inserting quality control samples in routine runs. The SEC coupled with a differential refractive-index detector (SEC/RI) was validated using a pre-study validation process in accordance with the ICH-Q2 (R1) guidelines and in-study monitoring in accordance with the Analytical Target Profile (ATP) criteria. The total error and ß-expectation tolerance interval rules were used to assess method suitability and control the risk of incorrectly accepting unsuitable analytical methods. The aggregation kinetics data were interpreted using a modified Lumry-Eyring model. The true order of the reaction was determined using the initial-rate approach. All the kinetic data show a linear Arrhenius dependence within the studied temperature range. The Arrhenius approach over-predicted the aggregation rate for 5°C, but provides an idea of the aggregation process and amount of aggregate formed. In any case, real-time stability data are necessary to establish the product shelf-life.

Bevacizumab clearance through the iridocorneal angle following intravitreal injection in a rat model.

Antivascular endothelial growth factor (Anti-VEGF) agents have been widely used for a variety of ocular disorders. The etiology of sustained ocular hypertension following intravitreal administration of anti-VEGF agents is yet to be unraveled. Our study investigates and characterizes the presence of intravitreally injected bevacizumab in the aqueous outflow channels of a rat model. Choroidal neovascularization (CNV) was induced by diode laser photocoagulation to the right eye of twelve Brown Norway rats. Bevacizumab (25 mg/ml) was injected intravitreally after 3 days. Immediately after bevacizumab injection, and 3, 6, 24 and 48 h later, animals were euthanized for immunofluorescence staining. Donkey anti-human IgG labeled with Alexa Fluor(®) 488 was used for bevacizumab immunoreactivity detection. Anti-CD31 antibody was used as a marker for Schlemm's canal endothelial cells. Untreated eyes were used as negative controls. The intensity of the immunostaining was analyzed qualitatively. Bevacizumab immunoreactivity was found in the aqueous outflow channels including the trabecular meshwork and Schlemm's canal immediately after injection, and declined incrementally within the following hours. Forty-eight hours after the injection, no bevacizumab staining was detected in the aqueous outflow channel structures. Our manuscript demonstrates the presence of bevacizumab in the trabecular meshwork and Schlemm's canal structures after intravitreal injection in a CNV induced rat model. Bevacizumab molecules passed through the aqueous outflow channels within 48 h after intravitreal bevacizumab injection.

In Vivo Imaging of Fluorescent Probes Linked to Antibodies Against Human and Rat Vascular Endothelial Growth Factor.

PURPOSE: Anti-VEGF therapy has improved functional outcome for many patients with neovascular AMD. A particular challenge in routine clinical application is to find the best treatment regimen as a high degree of interindividual variability of disease activity has been noted. The aim of the study was to investigate fluorescent probes linked to antibodies against VEGF for in vivo imaging in an animal model. METHODS: Bevacizumab, B20-4.1.1 and AF564 were covalently attached to the novel dye 6S-indocyanine green (ICG) maleimide. Binding and proliferation properties were assessed. In a rat model of laser-induced choroidal neovascularization, retinal uptake and topographic localization of antibody-conjugates were analyzed. Distribution and accumulation of the probes were determined by immunohistochemistry and flow cytometry analysis. RESULTS: Antibody-conjugates retained target binding affinity and showed no toxicity. In vivo imaging showed a strong fluorescence immediately following an intravenous or intravitreal injection. While accumulation within the laser lesions was visualized for all three antibody conjugates, the signal strength and the duration of fluorescence varied. In addition, distinct fluorescent spots were also recognized. Patterning and in-depth analyses including histology and flow cytometry data strongly suggest that the fluorescent spots represent labeled microglial cells and/or macrophages. CONCLUSIONS: Pharmacokinetics of fluorescent-labeled bevacizumab, B20-4.1.1 and AF564 can be investigated in vivo. In this model, interpretation of long-term in vivo observations is difficult because of a possible rat-specific immune response and challenges to image localized binding of soluble VEGF. Further investigations in a primate model and the use of appropriate antibodies directed against the VEGF-receptor may represent alternative approaches.