Sérgio Luiz Dalmora

Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization

BackgroundBiopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells.ResultsHere we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture.ConclusionThe recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.

Analysis of recombinant human growth hormone directly in osmotic shock fluids

An isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) method for the determination of human growth hormone (hGH) directly in osmotic shock fluids is described. This methodology allows an initial rapid evaluation of the quality and quantity of hGH being secreted in the bacterial periplasmic space right after, or even during fermentation. Considering that RP-HPLC does not identify size isomers, these were determined via a parallel run of the same osmotic shock fluid on high-performance size-exclusion chromatography, coupled with radioimmunoassay, of the eluted fractions. The methodology provides a complete picture, within 24 h from the beginning of the fermentation process, of the recombinant protein being produced with respect to its activity, identity, yield, and hGH-related contaminants. These latter include sulfoxide and desamido derivatives, dimer and high-molecular-mass forms.

Biological evaluation of recombinant human erythropoietin in pharmaceutical products

The potencies of mammalian cell-derived recombinant human erythropoietin pharmaceutical preparations, from a total of five manufacturers, were assessed by in vivo bioassay using standardized protocols. Eight-week-old normocythemic mice received a single subcutaneous injection followed by blood sampling 96 h later or multiple daily injections with blood sampling 24 h after the last injection. Reticulocyte counting by microscopic examination was employed as the end-point using the brilliant cresyl blue or selective hemolysis methods, together with automated flow cytometry. Different injection schedules were investigated and dose-response curves for the European Pharmacopoeia Biological Reference Preparation of erythropoietin were compared. Manual and automated methods of reticulocyte counting were correlated with respect to assay validity and precision. Using 8 mice per treatment group, intra-assay precision determined for all of the assays in the study showed coefficients of variation of 12.1-28.4% for the brilliant cresyl blue method, 14.1-30.8% for the selective hemolysis method and 8.5-19.7% for the flow cytometry method. Applying the single injection protocol, a combination of at least two independent assays was required to achieve the precision potency and confidence limits indicated by the manufacturers, while the multiple daily injection protocol yielded the same acceptable results within a single assay. Although the latter protocol using flow cytometry for reticulocyte counting gave more precise and reproducible results (intra-assay coefficients of variation: 5.9-14.2%), the well-characterized manual methods provide equally valid alternatives for the quality control of recombinant human erythropoietin therapeutic products.

Uncaria tomentosa-Adjuvant Treatment for Breast Cancer: Clinical Trial.

Breast cancer is the most frequent neoplasm affecting women worldwide. Some of the recommended treatments involve chemotherapy whose toxic effects include leukopenia and neutropenia. This study assessed the effectiveness of Uncaria tomentosa (Ut) in reducing the adverse effects of chemotherapy through a randomized clinical trial. Patients with Invasive Ductal Carcinoma—Stage II, who underwent a treatment regimen known as FAC (Fluorouracil, Doxorubicin, Cyclophosphamide), were divided into two groups: the UtCa received chemotherapy plus 300 mg dry Ut extract per day and the Ca group that only received chemotherapy and served as the control experiment. Blood samples were collected before each one of the six chemotherapy cycles and blood counts, immunological parameters, antioxidant enzymes, and oxidative stress were analyzed. Uncaria tomentosa reduced the neutropenia caused by chemotherapy and was also able to restore cellular DNA damage. We concluded that Ut is an effective adjuvant treatment for breast cancer.

Avaliação da atividade e caracterização de eritropoietina humana recombinante em produtos farmacêuticos

The identification of rhEPO in pharmaceutical products was carried out by polyacrylamide gel electrophoresis and detection with specific antibodies that revealed a typical single broad band similar to that obtained with the European Pharmacopoeia Biological Reference Preparation for erythropoietin. The isoelectric focusing with immunodetection revealed extensive heterogeneity with 5-6 isoforms, characteristic according to the manufacturer. The potency was assessed by the normocythaemic mouse assay with values within 67.6% and 119.4% calculated against the stated potency. The precision evaluated by the weight was within 200 and 389, for the combined assays. The toxicity, bacterial endotoxins, and pH gave variable results according to the batch. In conclusion, we emphasize the importance of the batch-to-batch tests and assays that would guarantee the quality and therapeutic efficacy of the pharmaceutical products.

Validation of an RP‐LC Method and Assessment of rhG‐CSF in Pharmaceutical Formulations by Liquid Chromatography and Biological Assay

Abstract Gradient reversed‐phase liquid chromatography (RP‐LC) was validated for the analysis of rhG‐CSF in pharmaceutical formulations. The LC method was carried out on a Jupiter C4 column (250 mm×4.6 mm I.D.), the mobile phase A consisted of water:acetonitrile (90:10, v/v) with 0.1% TFA and the mobile phase B was water:acetonitrile (20:80, v/v) with 0.1% TFA, run at a flow rate of 0.5 mL/min and detection at 280 nm. Validation parameters were evaluated and the method was linear in the range of 10–300 µg/mL. The dimers, high molecular mass forms, sulphoxides, and deamidates were analysed by the LC methods and then subjected to independent neutropenia mouse bioassay, giving overall biological activities within 13.47% and 15.63%. The pharmaceutical samples were analysed by the chromatographic methods and compared to the bioassay, showing mean difference between the estimated potency of 2.04% lower for the RP‐LC, and 4.03% lower for the SE‐LC, with significant correlation (P>0.05). Due to the bioactivity of the rhG‐CSF‐related proteins, the SE‐LC is proposed in combination with the RP‐LC as an alternative to the bioassay for the potency assessment, improving the quality control of rhG‐CSF in pharmaceutical dosage forms.

Assessment of rhEPO in Pharmaceutical Formulations by a Reversed‐Phase Liquid Chromatography Method and Bioassay

Abstract A gradient reversed‐phase liquid chromatography (RP‐LC) was developed for the analysis of alpha and beta rhEPO in pharmaceutical formulations. The RP‐LC method was carried out on a Jupiter C4 column (250 mm×4.6 mm I.D., with a pore size of 300 Å). The elution was performed by a gradient at a constant flow rate of 0.5 mL/min. Mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) and mobile phase B consisted of 0.08% TFA:acetonitrile (30∶70, v/v), using a photodiode array (PDA) detection at 280 nm. The chromatographic separation was obtained within 60 min and was linear in the concentration range of 10–150 µg/mL. The parameters validated, such as the specificity, precision, accuracy, and robustness gave results within the acceptable range. The pharmaceutical samples were analysed by the chromatographic method and compared to the normocythaemic mice bioassay, showing the mean difference between the estimated potencies of 11.2%±1.8 higher for the RP‐LC, with significant correlation (r=0.9799) as calculated by the Pearsons coefficient. The proposed RP‐LC method represents an alternative to the bioassay that can be applied for the potency assessment, improving the quality control of rhEPO in pharmaceutical dosage forms.

Determination of rupatadine in pharmaceutical formulations by a validated stability-indicating MEKC method

A stability-indicating MEKC was developed and validated for the analysis of rupatadine in tablet dosage forms, using nimesulide as internal standard. The MEKC method was performed on a fused-silica capillary (50 microm id; effective length, 40 cm). The BGE consisted of 15 mM borate buffer and 25 mM anionic detergent SDS solution at pH 10. The capillary temperature was maintained at 35 degrees C and the applied voltage was 25 kV. The injection was performed using the hydrodynamic mode at 50 mbar for 5 s, with detection by photodiode array detector set at 205 nm. The method was linear in the range of 0.5-150 microg/mL (r2=0.9996). The specificity and stability-indicating capability of the method were proven through degradation studies inclusive by MS, and showing also that there was no interference of the excipients and no increase of the cytotoxicity. The accuracy was 99.98% with bias lower than 1.06%. The LOD and LOQ were 0.1 and 0.5 microg/mL, respectively. The proposed method was successfully applied for the quantitative analysis of rupatadine in pharmaceutical formulations, and the results were compared to a validated RP-LC method, showing non-significant difference (p>0.05).

Analysis of human luteinizing hormone and human chorionic gonadotropin preparations of different origins by reversed-phase high-performance liquid chromatography

Specific reversed-phase high-performance liquid chromatography conditions are reported for the analysis of recombinant and native human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) preparations. Heterodimeric hLH, hCG and their alpha- and beta-subunits migrated with significantly different retention times (t(R)) in the following order of increasing hydrophobicity: alpha-hCG<alpha-hLH<hCG<hLH<beta-hCG<beta-hLH. Under these conditions, the main peak of three hCG preparations ran about 4% faster than the average t(R) (38.35+/-0.42 min; RSD=1.1%) of four hLH preparations. Four heterogeneous urinary products were also analyzed, hLH, hFSH and hCG peaks being identified. Quantitative analysis was validated for the homogeneous preparations and a highly linear dose-response curve (r=0.99998; p<0.0001; n=20) used to assess the accuracy, precision and sensitivity of the analysis. Quantification of the different gonadotropins in the heterogeneous preparations was also carried out, but with limitations in accuracy.

A pilot study on potency determination of human follicle-stimulating hormone: A comparison between reversed-phase high-performance liquid chromatography method and the in vivo bioassay

Reversed-phase high-performance liquid chromatography (RP-HPLC) was compared with the classical Steelman-Pohley bioassay (BA), based on animal use, for the determination of human follicle-stimulating hormone (hFSH) biological activity. A linear relationship (BA(IU)=0.9925 RP-HPLC(IU)-1.3165) with a highly significant correlation (r=0.9371; p<0.0001; n=24) was found for these two methods for six hFSH preparations of different origins. The mean difference between the bioactivity predicted from RP-HPLC data via this equation and the mean of the bioactivities obtained with the two methods for six other hFSH preparations was -1.4%, with a 95% confidence interval of -9.3 to +6.6%. The precision of these parameters was 1.63% and 2.82%, respectively. These results demonstrate that RP-HPLC is a viable physical-chemical alternative to the use of an in vivo bioassay for hFSH potency determination, applicable also to hFSH Standards containing large amounts of human serum albumin.