Assessment of pectin-coated magnetite nanoparticles in low-energy water desalination applications.

Novel magnetite nanoparticles (NPs) modified with pectin coating were fabricated, characterized, and evaluated as potential draw solute in a forward osmosis (FO) process for water desalination applications. The prepared NPs had a spherical shape with an average diameter of 200 nm and saturation magnetization of 23.13 emu/g. Thermogravimetric analysis (TGA) and FTIR spectra elucidated the successful pectin coating on magnetite surface. The potential use of the fabricated NPs in water desalination was conducted via a newly developed lab-scale FO system. Deionized water, saline water (0.2, 0.5, and 1 g% NaCl solution), and real well water (TDS = 0.9 g%) were used as feed solutions. In all experiments, the water flux gradually decreased along with the extension of experimental time and NaCl rejection rate by the FO membrane was measured to be higher than 95%. Moreover, it was found that the pectin-coated magnetite NPs demonstrated to be able to draw clean water across the FO membrane from well water with a remarkable salt rejection of 97%. Thus, it is believed that the proposed FO system using pectin-coated magnetite NPs as draw solute can be a promising technique for desalination of well waters in an environmental-friendly and energy-saving manner.

Multivariate Validated Models for Simultaneous Determination of Ibuprofen and Famotidine in the Presence of Related Substances.

Two multivariate validated spectrophotometric methods, namely partial least-squares (PLS) and principal component regression (PCR), were developed and validated for the determination of ibuprofen and famotidine in presence of famotidine degradation products and ibuprofen impurity (4-isobutylacetophenone). A calibration set was prepared in which the two drugs together with the degradation products and impurity were modeled using a multilevel multifactor design. This calibration set was used to build the PLS and PCR models. The proposed models successfully predicted the concentrations of both drugs in validation samples, with low root mean square error of cross validation (RMSECV) percentage. The method was validated by the estimate of the figures of merit depending on the net analyte signal. The results of the two models showed that the simultaneous determination of both drugs could be performed in the concentration ranges of 100-500 µg/mL for ibuprofen and 5-25 µg/mL for famotidine. The proposed multivariate calibration methods were applied for the determination of ibuprofen and famotidine in their pharmaceutical formulation, and the results were verified by the standard addition technique.

Comparative Assessment of the Effect of Hyper-glycosylation on the Pattern and Kinetics of Degradation of Darbepoetin Alfa using a Stability-Indicating Orthogonal Testing Protocol.

Darbepoetin alfa (DA); hyper-glycosylated Erythropoietin alfa (EPO) is an essential treatment of anemia in patients with chronic kidney failure and cancer. In this study, DA and EPO were subjected to physicochemical stress factors that might be encountered during production, transport and storage (pH, temperature, agitation, repeated freeze-thaw and oxidation). An orthogonal stability-indicating assay protocol comprised of SE-HPLC, RP-HPLC, ELISA and SDS-PAGE was developed and validated to investigate the effect of further glycosylation of DA on the pattern and kinetics of degradation. Results showed a relatively higher stability and lower tendency to form high molecular weight aggregates in the case of DA when compared to EPO, under equivalent stress conditions. Dimers and aggregates were formed for both drugs across the whole pH range and following incubation at temperatures higher than 2-8°C or repeated freeze/thaw. The same observation was noted upon agitation of standard samples prepared in the formulation buffers at high speed and upon oxidation with hydrogen peroxide. The agreement between SE-HPLC, supported with spectral purity data and ELISA confirmed the specificity of both techniques for the intact drugs. Results of RP-HPLC and SDS-PAGE indicated that dimerization occurred through disulfide and bi-tyrosine covalent bonds in the case of pH and oxidation, respectively. It was evident that aggregation was significantly suppressed upon increasing the glycan size and under any of the studied stress factors loss of the glycan has not been observed. These observations supported with the slow kinetics of degradation confirmed the superiority of glyco-engineering over chemical pegylation to enhance the stability of EPO. Formation of such potentially immunogenic product-related impurities at all tested stress factors confirmed the need for orthogonal testing protocols to investigate the complex pattern of degradation of such sensitive products.

Evaluation of the combined effects of pegylation and glycosylation on the stability of erythropoietin using a stability-indicating SE-HPLC.

Recombinant human erythropoietin (rhEPO) is a commonly used biopharmaceutical for the treatment of anemia-associated disorders. Epogen; glycosylated erythropoietin (G-EPO) has short half-life and poor stability. Pegylated Epogen (Peg-G-EPO) was introduced to the market to overcome these limitations. The combined effects of pegylation and glycosylation on the stability of Peg-G-EPO was studied. Determination of Peg-G-EPO in the presence of its degradation products was achieved using SE-HPLC. The assay was validated according to ICH guidelines over concentration range of 50.00-320.00 µg/mL (r 0.9999). A mobile phase of 50 mM phosphate buffer (pH 6.5) with 300 mM sodium chloride and 20% ethanol was employed. Isocratic elution was carried out at 0.5 mL/min over run time of 30 min. Peg-G-EPO was found stable towards mechanical agitation only at low concentrations while it was stable towards repeated freeze/thaw; regardless of the concentration. Effect of temperature and pH were also investigated and Peg-G-EPO was found stable within narrow ranges. Results indicated formation of small molecular weight and very high molecular weight aggregates that have been filtered-off the column. Although Peg-G-EPO was found relatively more stable than its non-pegylated but glycosylated version, results indicated the need for careful stability-assessment of Peg-G-EPO.

Zero and second-derivative synchronous fluorescence spectroscopy for the quantification of two non-classical ß-lactams in pharmaceutical vials: Application to stability studies.

The formation of metal chelates with various ligands may lead to the production of fluorescent chelates or enhance the fluorescence of the chelating agent. This paper describes two sensitive, selective and computer-solved methods, namely, zero order (SF) and second-derivative synchronous spectrofluorimetry (SDSFS) for nano-quantitation of two carbapenems; meropenem (MP) and ertapenem (EP). The methods are based on the chelation of MP with Tb3+ and EP with Zr4+ in buffered organic medium at pH 4.0 to produce fluorescent chelates. In the zero order method, the relative synchronous fluorescence intensity is measured at 327.0 nm at Δλ = 70.0 and 100.0 nm for MP and EP, respectively. The second method utilizes a second-derivative technique to enhance the method selectivity and emphasize a stability-indicating approach. The peak amplitudes (2 D) of the second-derivative synchronous spectra were estimated to be 333.06 and 330.06 nm for MP and EP, respectively. The proposed synchronous spectrofluorimetric methods were validated according to the International Conference on Harmonization (ICH) guidelines and applied successfully for the analysis of MP and EP in pure forms, pharmaceutical vials and in synthetic mixtures with different degradants of both drugs. Under optimum conditions, the mole-ratio method was applied and the co-ordination ratios of MP-Tb3+ and EP-Zr4+ chelates were found to be 1:1 and 1:3. The formation constants for the chelation complexes were evaluated using the Benesi-Hildebrand's equation; the free energy change (ΔG) was also calculated. The results indicated that EP-Zr4+ was more stable than the MP-Tb3+ chelate. Moreover, the developed methods were found to be selective and inexpensive for quantitative determination of both drugs in quality control laboratories at nano-levels.

Correlation between Dynamic Light Scattering and Size Exclusion High Performance Liquid Chromatography for Monitoring the Effect of pH on Stability of Biopharmaceuticals.

Aggregate formation is a major problem affecting both safety and efficacy of biopharmaceuticals and is associated with protein immunogenicity. Size exclusion high performance liquid chromatography (SE-HPLC) has always been the gold standard technique for detection and determination of protein aggregates. However, large protein aggregates may be filtered off and build up on top of the column leading to deterioration in column performance. Moreover, low-affinity protein aggregates may dissociate during analysis and thus not detected. On the other hand, dynamic light scattering (DLS) is a simple and non-destructive technique that can detect high molecular weight physical and chemical aggregates in their native environment. Here, three model biopharmaceutical proteins of different physicochemical properties were selected; quadrivalent human papillomavirus virus like particles vaccine (HPV VLP, physically assembled subunit vaccine, 55kDa), pegylated Interferon (PegIFN, pegylated non-glycosylated protein, 31.3kDa) and Pegylated Erythropoietin (PegEPO, pegylated and glycosylated protein, 60kDa). Samples were subjected to forced degradation conditions previously shown to lead to aggregate formation (pH 4.0, 8.0 and 10.0, at 37°C for 24h) and samples were analyzed using DLS and SE-HPLC. Generally, good agreement between the results of DLS and SE-HPLC was noted, regardless of the differences in physicochemical properties of the studied biopharmaceuticals. Results showed that aggregate formation was not detected in some cases by SE-HPLC and the decrease in the concentration of the monomeric forms indicated that such aggregates might have been filtered off the column. Although no single techniques can reveal all aspects of protein stability, DLS can serve as a screening tool to detect aggregate formation and cross-validate SE-HPLC results during batch release testing. Owing to its simplicity and low-sample volume requirements, DLS can be used even by hospital pharmacists to confirm absence of protein aggregates immediately before drug administration.

Development and validation of HPLC and CE methods for simultaneous determination of amlodipine and atorvastatin in the presence of their acidic degradation products in tablets.

Two methods were developed for separation and quantitation of amlodipine (AML) and atorvastatin (ATV) in the presence of their acidic degradation products. The first method was a simple isocratic RP-HPLC method while the second was capillary electrophoresis (CE). Degradation products were obtained by acidic hydrolysis of the two drugs and their structures were elucidated for the first time by IR and MS spectra. Degradation products did not interfere with the determination of either drug and the assays were therefore stability-indicating. The linearity of the proposed methods was established over the ranges 1-50 µg mL-1 for AML and ATV in the HPLC method and in the range of 3-50 and 4-50 µg mL-1 for AML and ATV, respectively, in the CE method. The proposed methods were validated according to ICH guidelines. The methods were successfully applied to estimation of AML and ATV in combined tablets.

Optimization and validation of ELISA immunoassay for the evaluation of in-vitro relative potency of a four-component human papillomavirus vaccine products.

A sandwich-type ELISA was optimized and validated to determine the in-vitro relative potency of the four-component prophylactic Human papillomavirus (HPV) vaccine. The vaccine contains the non-infectious virus like particles (VLP) corresponding to HPV Types 6, 11, 16 and 18. A modification of the desorption step required to release the VLPs from the aluminum adjuvant was carried out. Samples were incubated with citrate buffer for two hours at 37 °C instead of overnight incubation at room temperature. Assay validation was then carried out according to ICH guidelines. The assay was linear over a concentration range of 0.30-2000.00 ng/mL for the four HPV types. The assay was accurate and precise with a LOD of 0.092, 0.081, 0.086 and 0.068 ng/mL for type 6, 11, 16 and 18 respectively. Results were also statistically compared to those obtained using the reported ELISA assay and no significant difference was noted. In contrary to the reported ELISA protocol, this optimized immunoassay was superior with respect to analysis time, without affecting the accuracy and precision (RSD < 5%). This assay has proven to be useful for evaluating the efficacy of the quadrivalent HPV vaccine and is applicable for quality control and batch release purposes.

Immunoaffinity extraction using conformation-dependent antibodies coupled to SE-HPLC for the development of stability and potency-indicating assay for quadrivalent human papillomavirus vaccine.

Quadrivalent human papillomavirus (HPV) vaccine is formulated of four types of non-infectious recombinant virus like particles (VLPs) that are structurally and immunologically similar to the corresponding infectious HPV virus types 6, 11, 16 and 18. With almost identical physical, chemical and structural properties of the four types of VLPs, ELISA remains the only approved in vitro potency testing assay. In this study, an alternative industry-friendly, stability- and potency-indicating assay protocol was developed and validated for the determination of HPV vaccine. Vacuum-driven immunoaffinity extraction (IAE) was employed using type-specific, conformation-dependent antibodies against each type of HPV VLPs. ELISA assay was employed to evaluate the ability of IAE columns to specifically separate each of the four types of VLPs from their quadrivalent mixture. Mean percentage recoveries of 76.76±2.69, 69.12±5.79, 84.86±5.25 and 71.14±4.50% were obtained for VLPs types 6, 11, 16 and 18, respectively with no significant interference in each case. Antigen content was then determined using SE-HPLC over a concentration range of 5.00-20.00µg/mL (r>0.998) for VLPs type 6, 11, 16 and 18, respectively. The SE-HPLC assay was found accurate and precise (RSD<10.00%) with LOD ranging from 1.23-3.85µg/mL. The assay protocol was found superior to conventional ELISA assay with respect to simplicity, total analysis time and cost. Good correlation between the results of analysis obtained using IAE-SE-HPLC and ELISA demonstrated the suitability of the suggested assay protocol for stability and potency assessment with a good potential for implementation for batch release. This approach should be applicable for quality assessment of other vaccine preparations based on VLPs.

Comparative study between recent methods manipulating ratio spectra and classical methods based on two-wavelength selection for the determination of binary mixture of antazoline hydrochloride and tetryzoline hydrochloride.

A comparative study was developed between two classical spectrophotometric methods (dual wavelength method and Vierordt's method) and two recent methods manipulating ratio spectra (ratio difference method and first derivative of ratio spectra method) for simultaneous determination of Antazoline hydrochloride (AN) and Tetryzoline hydrochloride (TZ) in their combined pharmaceutical formulation and in the presence of benzalkonium chloride as a preservative without preliminary separation. The dual wavelength method depends on choosing two wavelengths for each drug in a way so that the difference in absorbance at those two wavelengths is zero for the other drug. While Vierordt's method, is based upon measuring the absorbance and the absorptivity values of the two drugs at their λ(max) (248.0 and 219.0 nm for AN and TZ, respectively), followed by substitution in the corresponding Vierordt's equation. Recent methods manipulating ratio spectra depend on either measuring the difference in amplitudes of ratio spectra between 255.5 and 269.5 nm for AN and 220.0 and 273.0 nm for TZ in case of ratio difference method or computing first derivative of the ratio spectra for each drug then measuring the peak amplitude at 250.0 nm for AN and at 224.0 nm for TZ in case of first derivative of ratio spectrophotometry. The specificity of the developed methods was investigated by analyzing different laboratory prepared mixtures of the two drugs. All methods were applied successfully for the determination of the selected drugs in their combined dosage form proving that the classical spectrophotometric methods can still be used successfully in analysis of binary mixture using minimal data manipulation rather than recent methods which require relatively more steps. Furthermore, validation of the proposed methods was performed according to ICH guidelines; accuracy, precision and repeatability are found to be within the acceptable limits. Statistical studies showed that the methods can be competitively applied in quality control laboratories.

Advanced stability indicating chemometric methods for quantitation of amlodipine and atorvastatin in their quinary mixture with acidic degradation products.

Two advanced, accurate and precise chemometric methods are developed for the simultaneous determination of amlodipine besylate (AML) and atorvastatin calcium (ATV) in the presence of their acidic degradation products in tablet dosage forms. The first method was Partial Least Squares (PLS-1) and the second was Artificial Neural Networks (ANN). PLS was compared to ANN models with and without variable selection procedure (genetic algorithm (GA)). For proper analysis, a 5-factor 5-level experimental design was established resulting in 25 mixtures containing different ratios of the interfering species. Fifteen mixtures were used as calibration set and the other ten mixtures were used as validation set to validate the prediction ability of the suggested models. The proposed methods were successfully applied to the analysis of pharmaceutical tablets containing AML and ATV. The methods indicated the ability of the mentioned models to solve the highly overlapped spectra of the quinary mixture, yet using inexpensive and easy to handle instruments like the UV-VIS spectrophotometer.

Mean centering of double divisor ratio spectra, a novel spectrophotometric method for analysis of ternary mixtures.

A novel spectrophotometric method was developed for determination of ternary mixtures without previous separation, showing significant advantages over conventional methods. The new method is based on mean centering of double divisor ratio spectra. The mathematical explanation of the procedure is illustrated. The method was evaluated by determination of model ternary mixture and by the determination of Amlodipine (AML), Aliskiren (ALI) and Hydrochlorothiazide (HCT) in laboratory prepared mixtures and in a commercial pharmaceutical preparation. For proper presentation of the advantages and applicability of the new method, a comparative study was established between the new mean centering of double divisor ratio spectra (MCDD) and two similar methods used for analysis of ternary mixtures, namely mean centering (MC) and double divisor of ratio spectra-derivative spectrophotometry (DDRS-DS). The method was also compared with a reported one for analysis of the pharmaceutical preparation. The method was validated according to the ICH guidelines and accuracy, precision, repeatability and robustness were found to be within the acceptable limits.

Strategy Approach for Direct Enantioseparation of Hyoscyamine Sulfate and Zopiclone on a Chiral αl-Acid Glycoprotein Column and Determination of Their Eutomers: Thermodynamic Study of Complexation.

Rapid and simple isocratic high-performance liquid chromatographic methods with UV detection were developed and validated for the direct resolution of racemic mixtures of hyoscyamine sulfate and zopiclone. The method involved the use of αl -acid glycoprotein (AGP) as chiral stationary phase. The stereochemical separation factor (ά) and the stereochemical resolution factor (Rs ) obtained were 1.29 and 1.60 for hyoscyamine sulfate and 1.47 and 2.45 for zopiclone, respectively. The method was used for determination of chiral switching (eutomer) isomers: S-hyoscyamine sulfate and eszopiclone. Several mobile phase parameters were investigated for controlling enantioselective retention and resolution on the chiral AGP column. The influence of mobile phase, concentration and type of uncharged organic modifier, ionic strength, and column temperature on enantioselectivity were studied. Calibration curves were linear in the ranges of 1-10 µg mL(-1) and 0.5-5 µg mL(-1) for S-hyoscyamine sulfate and eszopiclone, respectively. The method is specific and sensitive, with lower limits of detection and quantifications of 0.156, 0.515 and 0.106, 0.349 for S-hyoscyamine sulfate and eszopiclone, respectively. The method was used to identify quantitatively the enantiomers profile of the racemic mixtures of the studied drugs in their pharmaceutical preparations. Thermodynamic studies were performed to calculate the enthalpic ΔH and entropic ΔS terms. The results showed that enantiomer separation of the studied drugs were an enthalpic process.

Quality in the pharmaceutical industry - A literature review.

OBJECTIVES: The aim of this study is to:a.Highlight the most important guidelines and practices of quality in the pharmaceutical industry.b.Organize such guidelines and practices to create a guide to pave the way for other researchers who would like to dig deeper into these guidelines and practices. DESIGN: A review was conducted of 102 publications; 56 publications were concerned with the pharmaceutical quality directly while 46 publications were concerned with the general quality practices. The content of those sources was analyzed and the following themes were identified:a.Research theme 1: Guidelines of the pharmaceutical quality.b.Research theme 2: General practices recently applied in the pharmaceutical industry. MAIN OUTCOME MEASURES: The following guidelines were identified and reviewed: WHO guidelines, FDA guidelines, EU guidelines and ICH guidelines in the research theme I. In research theme II; the following topics were identified and reviewed: quality risk management, quality by design, corrective actions and preventive actions, process capability analysis, Six Sigma, process analytical technology, lean manufacturing, total quality management, ISO series and HACCP. RESULTS: Upon reviewing the previously highlighted guidelines and the practices that are widely applied in the pharmaceutical industry, it was noticed that there is an abundant number of papers and articles that explain the general guidelines and practices but the literature lack those describing application; case studies of the pharmaceutical factories applying those guidelines and significance of those guidelines and practices. CONCLUSIONS: It is recommended that the literature would invest more in the area of application and significance of guidelines and practices. New case studies should be done to prove the feasibility of such practices.

Continuous Wavelet Transform, a powerful alternative to Derivative Spectrophotometry in analysis of binary and ternary mixtures: A comparative study.

A comparative study was established between two signal processing techniques showing the theoretical algorithm for each method and making a comparison between them to indicate the advantages and limitations. The methods under study are Numerical Differentiation (ND) and Continuous Wavelet Transform (CWT). These methods were studied as spectrophotometric resolution tools for simultaneous analysis of binary and ternary mixtures. To present the comparison, the two methods were applied for the resolution of Bisoprolol (BIS) and Hydrochlorothiazide (HCT) in their binary mixture and for the analysis of Amlodipine (AML), Aliskiren (ALI) and Hydrochlorothiazide (HCT) as an example for ternary mixtures. By comparing the results in laboratory prepared mixtures, it was proven that CWT technique is more efficient and advantageous in analysis of mixtures with severe overlapped spectra than ND. The CWT was applied for quantitative determination of the drugs in their pharmaceutical formulations and validated according to the ICH guidelines where accuracy, precision, repeatability and robustness were found to be within the acceptable limit.

Development and validation of chromatographic methods for simultaneous determination of ibuprofen and famotidine in presence of related substances in pharmaceutical formulations.

Two validated methods for the simultaneous determination of ibuprofen and famotidine in the presence of ibuprofen impurity (4-isobutylacetophenone) and or famotidine degradation products were described. The first method was a simple TLC method where separation was performed on silica gel platesusing ethyl acetate: methanol: ammonia (9:2:1, by volume) as a mobile phase. Rf values were found to be 0.40, 0.94, 0.66, 0.27, 0.83 for ibuprofen, 4-isobutylacetophenone, famotidine, famotidine acid and basic degradation products, respectively. The second method is by HPLC on C18 column using methanol: phosphate buffer pH 3 (80:20, v/v) as a mobile phase. Retention times were found to be 2.2, 9.9, and 8.6 for famotidine, ibuprofen, and 4-isobutylacetophenone, respectively. Both methods were validated according to the ICH guidelines and applied for the determination of the two drugs in pure powder and combined dosage form without interference from the excipients.

Application of the ratio difference spectrophotometry to the determination of ibuprofen and famotidine in their combined dosage form: comparison with previously published spectrophotometric methods.

Ratio difference spectrophotometric method was developed for the determination of ibuprofen and famotidine in their mixture form. Ibuprofen and famotidine were determined in the presence of each other by the ratio difference spectrophotometric (RD) method where linearity was obtained from 50 to 600µg/mL and 2.5 to 25µg/mL for ibuprofen and famotidine, respectively. The suggested method was validated according to ICH guidelines and successfully applied for the analysis of ibuprofen and famotidine in their pharmaceutical dosage forms without interference from any additives or excipients.

Different signal processing techniques of ratio spectra for spectrophotometric resolution of binary mixture of bisoprolol and hydrochlorothiazide; a comparative study.

Five signal processing techniques were applied to ratio spectra for quantitative determination of bisoprolol (BIS) and hydrochlorothiazide (HCT) in their binary mixture. The proposed techniques are Numerical Differentiation of Ratio Spectra (ND-RS), Savitsky-Golay of Ratio Spectra (SG-RS), Continuous Wavelet Transform of Ratio Spectra (CWT-RS), Mean Centering of Ratio Spectra (MC-RS) and Discrete Fourier Transform of Ratio Spectra (DFT-RS). The linearity of the proposed methods was investigated in the range of 2-40 and 1-22 µg/mL for BIS and HCT, respectively. The proposed methods were applied successfully for the determination of the drugs in laboratory prepared mixtures and in commercial pharmaceutical preparations and standard deviation was less than 1.5. The five signal processing techniques were compared to each other and validated according to the ICH guidelines and accuracy, precision, repeatability and robustness were found to be within the acceptable limit.

Validated green high-performance liquid chromatographic methods for the determination of coformulated pharmaceuticals: a comparison with reported conventional methods.

The introduction of sustainable development concepts to analytical laboratories has recently gained interest, however, most conventional high-performance liquid chromatography methods do not consider either the effect of the used chemicals or the amount of produced waste on the environment. The aim of this work was to prove that conventional methods can be replaced by greener ones with the same analytical parameters. The suggested methods were designed so that they neither use nor produce harmful chemicals and produce minimum waste to be used in routine analysis without harming the environment. This was achieved by using green mobile phases and short run times. Four mixtures were chosen as models for this study; clidinium bromide/chlordiazepoxide hydrochloride, phenobarbitone/pipenzolate bromide, mebeverine hydrochloride/sulpiride, and chlorphenoxamine hydrochloride/caffeine/8-chlorotheophylline either in their bulk powder or in their dosage forms. The methods were validated with respect to linearity, precision, accuracy, system suitability, and robustness. The developed methods were compared to the reported conventional high-performance liquid chromatography methods regarding their greenness profile. The suggested methods were found to be greener and more time- and solvent-saving than the reported ones; hence they can be used for routine analysis of the studied mixtures without harming the environment.

Chromatographic and electrophoretic assessment of Filgrastim biosimilars in pharmaceutical formulations.

An orthogonal testing protocol was developed and validated to assess the quality of Filgrastim biosimilars. Results were compared to those obtained from the innovator product. Initial screening was carried out using reducing and non-reducing gel electrophoresis. RP-LC was employed for the determination of Filgrastim in the presence of its oxidative degradation products. SEC and CIEF were used under non-denaturing conditions to reveal high molecular weight and charged impurities, respectively. RP-LC assay was found accurate (99.78±0.89) and precise over a linear concentration range of 9.38-300.00µg/ml with a LOD of 8.26µg/ml (0.44mM). SEC was carried out over a molecular weight range of 5.0-150.0kDa. CIEF was optimized using neutrally coated capillaries over a wide-range pH gradient (pH 3.0-10.0). Differences between the studied products were revealed using all these techniques. Impurities above the acceptable limits were detected in both biosimilar products. CIEF revealed heterogeneity in the active ingredient that has not been investigated by the manufacturers. Correlation of the obtained results indicated the presence of not only product-related impurities, but also process-related impurities. Results confirmed the need for in-house validated orthogonal testing protocols to be developed by local regulatory authorities. This should prevent access of substandard biosimilars to price-sensitive markets.