Changes in antibody binding and functionality after humanizing a murine scFv anti-IFN-α2: From in silico studies to experimental analysis.

The structural and dynamic changes introduced during antibody humanization continue to be a topic open to new contributions. For this reason, the study of structural and functional changes of a murine scFv (mu.scFv) anti-rhIFN-α2b after humanization was carried out.

Large Area Microfluidic Bioreactor for Production of Recombinant Protein.

To produce innovative biopharmaceuticals, highly flexible, adaptable, robust, and affordable bioprocess platforms for bioreactors are essential. In this article, we describe the development of a large-area microfluidic bioreactor (LM bioreactor) for mammalian cell culture that works at laminar flow and perfusion conditions. The 184 cm 32 cisterns LM bioreactor is the largest polydimethylsiloxane (PDMS) microfluidic device fabricated by photopolymer flexographic master mold methodology, reaching a final volume of 2.

The glycosylation of anti-rhIFN-α2b recombinant antibodies influences the antigen-neutralizing activity.

Objectives: The influence of glycosylation on the antigen-neutralizing ability of two potential biotherapeutic anti-human IFN-α2b antibodies composed by murine and humanized single-chain Fv fused to human Fcγ1 (chimeric and humanized scFv-Fc, respectively) was studied.

Results: Chimeric antibodies produced in CHO-K1 and HEK293 mammalian cells showed no differences in the antigen-antibody affinity but demonstrated differences in the in vitro neutralization of IFN-α2b activity. On the other hand, the humanized antibodies produced in the same cell types showed differences in both the antigen-antibody affinity and the antigen-neutralizing ability.

Design and validation of an immuno-PCR assay for IFN-α2b quantification in human plasma.

Nowadays, IFN-α is considered a promising therapeutic target for systemic lupus erythematosus. An immuno-PCR (iPCR) was developed to quantify low amounts of IFN-α in human plasma followed by a deep analysis of the methodologic robustness throughout quality by design approach. An accurate, sensitive, selective and versatile iPCR was validated.

Production of monoclonal antibodies in microfluidic devices.

Herein, a microfluidic device with cistern design for cultivation of adherent eukaryotic cells for the production of recombinant proteins is presented. The geometric configuration of the microchannels in the device provided laminar flow with reduced velocity profiles in the cisterns, resulting in an adequate microenvironment for long-term adherent cell growth with passive pumping flow cycles of 24 hours. CHO-ahIFNα2b and HEK-ahIFNα2b adherent cell lines expressing a novel anti-hIFN-α2b recombinant monoclonal antibody (MAb) for the treatment of systemic lupus erythematosus were cultured on the surface of PDMS/glass microchannels coated with poly-d-lysine.

An unusual cysteine V87 affects the antibody fragment conformations without interfering with the disulfide bond formation.

The Cys residues are almost perfectly conserved in all antibodies. They contribute significantly to the antibody fragment stability. The relevance of two natural contiguous Cys residues of an anti-recombinant human-follicle stimulation hormone (rhFSH) in a format of single-chain variable fragment (scFv) was studied.

A highly efficient modified human serum albumin signal peptide to secrete proteins in cells derived from different mammalian species.

Signal peptides (SPs) are key elements in the production of recombinant proteins; however, little information is available concerning different SP in mammalian cells other than CHO. In order to study the efficiency of different SPs to direct the traffic along the secretory pathway of the green fluorescence protein (GFP) and a scFv-Fc fusion protein; CHO-K1, HEK293 and NS0 cell lines were transfected in a transient and stable way. SP of human azurocidin (AZ), modified human albumin (mSA), modified Cricetulus griseus Ig kappa chain V III region MOPC 63 like (mIgκ C) and modified human Ig kappa chain V III region VG (mIgκ H) were evaluated.

Plants contain two SCO proteins that are differentially involved in cytochrome c oxidase function and copper and redox homeostasis.

Two Arabidopsis thaliana genes (HCC1 and HCC2), resulting from a duplication that took place before the emergence of flowering plants, encode proteins with homology to the SCO proteins involved in copper insertion during cytochrome c oxidase (COX) assembly in other organisms. Heterozygote HCC1 mutant plants produce 25% abnormal seeds with defective embryos arrested at the heart or torpedo stage. These embryos lack COX activity, suggesting that the requirement of HCC1 during the early stages of plant development is related with its COX assembly function.

Characterization of Arabidopsis thaliana genes encoding functional homologues of the yeast metal chaperone Cox19p, involved in cytochrome c oxidase biogenesis.

The Arabidopsis thaliana genome contains two nearly identical genes which encode proteins showing similarity with the yeast metal chaperone Cox19p, involved in cytochrome c oxidase biogenesis. One of these genes (AtCOX19-1) produces two transcript forms that arise from an alternative splicing event and encode proteins with different N-terminal portions. Both AtCOX19 isoforms are imported into mitochondria in vitro and are found attached to the inner membrane facing the intermembrane space.

Transcriptional coordination of the biogenesis of the oxidative phosphorylation machinery in plants.

Publicly available microarray experiments were used to analyze Arabidopsis thaliana genes whose expression is correlated with that of nuclear genes encoding components of the oxidative phosphorylation machinery (OxPhos genes). This analysis indicated the existence of coordination in the expression of genes encoding components of the five respiratory complexes. For these genes, preferential expression was observed in anthers and roots, especially in the elongation zone, while reduced or very low relative expression was evident in leaves and mature pollen grains.