Extensive preclinical evaluation of an infliximab biosimilar candidate.

Infliximab is therapeutic monoclonal antibody (mAb) against TNF-α employed in the treatment of immunoinflammatory diseases. The development of biosimilar mAbs is a global strategy to increase drug accessibility and reduce therapy-associated costs. Herein we compared key physicochemical characteristics and biological activities produced by infliximab and infliximab-Probiomed in order to identify functionally relevant differences between the mAbs. Binding of infliximab-Probiomed to TNF-α was specific and had kinetics comparable to that of the reference product. Both mAbs had highly similar neutralizing efficacy in HUVEC cell cultures stimulated with TNF-α. In vitro induction of CDC and ADCC were also similar between the evaluated products. In vivo comparability was assessed using a transgenic mouse model of arthritis that expresses human TNF-α in a 13-week multiple-administration study. Infliximab and infliximab-Probiomed showed comparable efficacy, safety, and pharmacokinetic profiles. Our results indicate that infliximab-Probiomed has highly similar activities to infliximab in preclinical models, warranting a clinical evaluation of its biosimilarity.

Design of a strong cation exchange methodology for the evaluation of charge heterogeneity in glatiramer acetate.

Complex pharmaceuticals are in demand of competent analytical methods able to analyze charge heterogeneity as a critical quality attribute (CQA), in compliance with current regulatory expectations. A notorious example is glatiramer acetate (GA), a complex polypeptide mixture useful for the treatment of relapsing-remitting multiple sclerosis. This pharmaceutical challenges the current state of analytical technology in terms of the capacity to study their constituent species. Thus, a strong cation exchange methodology was designed under the lifecycle approach to support the establishment of GA identity, trough the evaluation of its chromatographic profile, which acts as a charge heterogeneity fingerprint. In this regard, a maximum relative margin of error of 5% for relative retention time and symmetry factor were proposed for the analytical target profile. The methodology met the proposed requirements after precision and specificity tests results, the former comprised of sensitivity and selectivity. Subsequently, method validation was conducted and showed that the method is able to differentiate between intact GA and heterogeneity profiles coming from stressed, fractioned or process-modified samples. In summary, these results provide evidence that the method is adequate to assess charge heterogeneity as a CQA of this complex pharmaceutical.

Phthalates and bisphenols migration in Mexican food cans and plastic food containers.

The presence of endocrine disruptors bisphenol-A, bisphenol-A-dimethacrylate, bisphenol-A-diglycidyl-ether, phthalic-acid, dibutyl-phthalate, diethyl-phthalate and dioctyl-phthalate was determined in vegetable cans, baby bottles and microwaveable containers from the Mexican market. Gas-Chromatography-Mass-Spectrometry was used for the identification and High-Performance-Liquid-Chromatography with UV/Visible light and fluorescence detectors was used for the quantification. Endocrine disruptors were found in all samples. PA and DOP were the substances most commonly found, and maximum concentrations were 9.549 and 0.664 µg/kg, respectively from a jalapeno peppers can. Bisphenol A, phthalic-acid, bisphenol-A-dimethacrylate, bisphenol-A-diglycidyl-ether, dioctyl-phtalate and dibutyl-phthalate were found in baby bottles and microwaveable containers.

Interferon-alpha 2b quantification in inclusion bodies using reversed phase-ultra performance liquid chromatography (RP-UPLC).

Interferon-alpha 2b (IFN-alpha 2b) is a recombinant therapeutic cytokine produced as inclusion bodies using a strain of Escherichia coli as expression system. After fermentation and recovery, it is necessary to know the amount of recombinant IFN-alpha 2b, in order to determine the yield and the load for solubilization, and chromatographic protein purification steps. The present work details the validation of a new short run-time and fast sample-preparation method to quantify IFN-alpha 2b in inclusion bodies using Reversed Phase-Ultra Performance Liquid Chromatography (RP-UPLC). The developed method demonstrated an accuracy of 100.28%; the relative standard deviations for method precision, repeatability and inter-day precision tests were found to be 0.57%, 1.54% and 1.83%, respectively. Linearity of the method was assessed in the range of concentrations from 0.05 mg/mL to 0.5 mg/mL, the curve obtained had a determination coefficient (r(2)) of 0.9989. Detection and quantification limits were found to be 0.008 mg/mL and 0.025 mg/mL, respectively. The method also demonstrated robustness for changes in column temperature, and specificity against host proteins and other recombinant protein expressed in the same E. coli strain.

Modified penicillin acylase signal peptide allows the periplasmic production of soluble human interferon-gamma but not of soluble human interleukin-2 by the Tat pathway in Escherichia coli.

Production of periplasmic human interferon-gamma (hINF-gamma) and human interleukin-2 (hIL-2) by the Tat translocation pathway in Escherichia coli BL21-SI was evaluated. The expression was obtained using the pEMR vector which contains the Tat-dependent modified penicillin acylase signal peptide (mSPpac) driven by the T7 promoter. The mSPpac-hINF-gamma was processed and the protein was transported to periplasm. Up to 30.1% of hINF-gamma was found in the periplasmic soluble fraction, whereas only 15% of the mSPpac-hIL-2 was processed, but hIL-2 was not found in the periplasmic soluble fraction.