Isolation and characterization of charge variants of infliximab biosimilar HS626.

Charge variants are the most commonly observed sources of heterogeneity in the routine manufacturing of monoclonal antibodies. To gain further insight into the structural foundation of charge heterogeneity and its influence on biological functions, an infliximab biosimilar HS626 from a biopharmaceutical facility was isolated by semipreparative cation exchange chromatography (CEX) to obtain fractions of acidic and basic charge variants and determine the main species. It was assessed again by CEX to ensure purities. Through a series of structural and physicochemical characterizations, we concluded that the acidic variants were caused by fragments, Met oxidation, Asn deamidation, higher levels of sialylation and galactosylation of N-linked glycans, and less high mannose. The basic variants resulted mainly from aggregates, fragments, and Met oxidation. Through further analysis of antigen binding affinity, cell death inhibitory activity, ADCC, and CDC, as well as FcRn, FcγRIIIa, and C1q affinity, we demonstrated that the charge heterogeneity did not affect biological functions. This research enhances the understanding of charge variants, which are usually effective components that should not be intentionally reduced unless biological functions are affected.

Influence of PPCPs on the performance of intermittently operated slow sand filters for household water purification.

Removal of pharmaceuticals and personal care products (PPCPs) from drinking water is usually enhanced by advanced oxidation which is not affordable in low income countries. Slow sand filtration has been found to be capable of removing anti-inflammatory compounds, and its low maintenance costs and easy operation make it an attractive technology for treating drinking water in many parts of the world. In addition, slow sand filters can be used at both large and household scales. The biofilm (i.e. schmutzdecke) developed on the top of the sand and within the upper layers of the sand is acknowledged to be responsible for the water purification. However, it is possible that the PPCPs may affect the schmutzdecke development and microbial community within the filters, and consequently the performance of the filter. This study investigated two household slow sand filters (for water purification) operated intermittently with and without contamination by six PPCPs. Eleven parameters were monitored in the affluent and effluent water, including bacterial species present and schmutzdecke biomass development. Results demonstrated that the household slow sand filter performance was not affected by the 2µgL-1 of PPCPs in the water. There was no significant difference between filters for total coliforms and E. coli removal, but there was considerable difference between sampling times. Biomass considerably increased with the number of filtrations in both filters and there was no significant difference between filter biomass. However, it was found that more bacterial species were present in the period with no contamination than during the contamination period. Bacillus anthracis and Exiguobacterium sp. showed to be resistant to the effects of the PPCPs. These suggest there are effects of PPCPs on bacterial species within the filter. However, the effect of the PPCPs on biomass was not conclusive in this study and needs to be further investigated.

Expression, purification and biological activity assessment of romiplostim biosimilar peptibody.

BACKGROUND: Romiplostim is a peptibody analogue of thrombopoietin (TPO) which regulates platelet production. This molecule consists of two main parts: Peptide sequences which like wild type TPO, mimics stimulation of TPO receptor and IgG1Fc, (Peptide + Antibody = Peptibody). This drug is used in treatment of chronic Immune Thrombocytopenic Purpura (ITP). METHODS: In this project E. coli bacteria were transformed by a construct harboring peptibody fusion gene. This construct consisted of two repeated peptide sequences which have fused to Carboxyl group of IgG1Fc. Designed construct in E. coli host resulted in protein expression in cytoplasm as inclusion body. The inclusion bodies were separated, washed and after denaturation and solubilization, in the last stage the desired peptibodies were refolded and purified. The resulting peptibodies were characterized by SDS-PAGE and Western immunoblotting. The bioactivity were assessed in vivo using subcutaneous injection in mice. RESULTS: Results showed accurate molecules were produced and purified. Also, in vivo experiment showed significant increment (more than two fold) of platelets compared to control group. CONCLUSION: In this study laboratory scale production of recombinant romiplostim showed proper in-vivo bioactivity. This new approach in expression and purification of this recently introduced thrombopoietin receptor agonist drug may be followed by scale up of its production to response the chronic ITP patient's demand.

Quality by Design (QbD)-Based Process Development for Purification of a Biotherapeutic.

Quality by Design (QbD) is currently receiving increased attention from the pharmaceutical community. As a result, most major biotech manufacturers are in varying stages of implementing QbD. Here, I present a case study that illustrates the step-by-step development using QbD of a purification process for the production of a biosimilar product: granulocyte colony-stimulating factor (GCSF). I also highlight and discuss the advantages that QbD-based process development offers over traditional approaches. The case study is intended to help those who wish to implement QbD towards the development and commercialization of biotech products.

Efficacy behind activity--phytotherapeutics are not different from pharmaceuticals.

CONTEXT: When evaluating bioactivity, efficacy, and toxicology of plant products, attention should be paid on pre-clinical versus clinical research. OBJECTIVE: To emphasize an evidence-based approach in the field of phytotherapy research. METHODS: The pyramid of scientific evidence was used in order to assess the quality of phytotherapy studies. RESULTS AND CONCLUSION: In terms of evidence-based phytotherapy, a step-by-step approach, ascending the "pyramid" of scientific evidence, is essential in order to collect correct information useful to clinicians and physicians, leant on the robust foundations of in vitro/in vivo studies.