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.

Recombinant human follicle stimulating hormone purification by a short peptide affinity chromatography.

Peptide KVPLITVSKAK was selected to design a synthetic ligand for affinity chromatography purification of recombinant human follicle stimulating hormone (rhFSH), based on the interaction of the hormone with the exoloop 3 of its receptor. The peptide was acetylated to improve its stability to degradation by exopeptidases. A cysteine was incorporated at the C-termini to facilitate its immobilization to the chromatographic activated SulfoLink agarose resin.

Single step recombinant human growth hormone (rhGH) purification from milk by peptide affinity chromatography.

Recombinant human growth hormone (rhGH) is used for the treatment of several pathologies, most of them related to growth. Although different expression systems can be used for its production, the milk from transgenic cows is one of the most interesting due to the high rhGH level achieved (5 g/L). We have designed and synthesized short peptides (9 or 10 amino acid long) using Fmoc chemistry and studied their ability to purify rhGH from milk once immobilized on an agarose support.

Alternating flow filtration as an alternative to internal spin filter based perfusion process: Impact on productivity and product quality.

This article reports the results obtained from comparison of internal spin filter (ISF) and alternating flow filtration (ATF) as cell retention systems, regarding cell growth, volumetric perfusion rate, cell specific perfusion rate and cell productivity in the fermentation process. As expected we were able to reach higher cell densities and to achieve longer runs since ATF systems are known to be less affected by fouling. Volumetric production of the reactor using the ATF system was 50-70% higher than the production achieved using the ISF due to higher cell density and a two-fold increase in the perfusion rate.

Glycosylation and antiproliferative activity of hyperglycosylated IFN-α2 potentiate HEK293 cells as biofactories.

Both CHO and HEK cells are interesting hosts for the production of biotherapeutics due to their ability to introduce post-translational modifications such as glycosylation. Even though oligosaccharide structures attached to proteins are conserved among eukaryotes, many differences have been found between therapeutic glycoproteins expressed in hamster and human derived cells. In this work, a hyperglycosylated IFN-α2b mutein (IFN4N) was produced in CHO and HEK cell lines and an extensive characterization of their properties was performed.

Establishment of a design space for biopharmaceutical purification processes using DoE.

Recent trends in the pharmaceutical sector are changing the way protein purification processes are designed and executed, moving from operating the process in a fixed point to allowing a permissible region in the operating space known as design space. This trend is driving product development to design quality into the manufacturing process (Quality by Design) and not to rely exclusively on testing quality in the product. A typical purification step has numerous operating parameters that can impact its performance.

Statistical optimization of influenza H1N1 production from batch cultures of suspension Vero cells (sVero).

Efficient vaccine production requires the growth of large quantities of virus produced with high yield from a safe host system. Human influenza vaccines are produced in embryonated chicken eggs. However, over the last decade many efforts have allowed the establishment of cell culture-derived vaccines.

Isolation and characterization of a subset of erythropoietin glycoforms with cytoprotective but minimal erythropoietic activity.

Although historically used for the treatment of anemia, erythropoietin (EPO) has emerged as a neurotrophic and neuroprotective agent in different conditions of neuronal damage (traumatic brain injury, ischemia, spinal cord compression, peripheral neuropathy, retinal damage, epilepsy, Parkinson's Disease, among others). Nonetheless, EPO's therapeutic application is limited due to its hematological side-effects. With the aim of obtaining EPO derivatives resembling the hormone isolated from cells and tissues of neural origin, a novel combination of less acidic EPO glycoforms -designated as neuroepoetin (rhNEPO)- was purified to homogeneity from the supernatant of a CHO-producing cell line by a four-step chromatographic procedure.

Determination of robustness and optimal work conditions for a purification process of a therapeutic recombinant protein using response surface methodology.

A typical chromatographic purification step has numerous operating parameters that can impact its performance. As it is not feasible to evaluate the influence of each one, the current practice in biopharmaceutical industry is to apply risk analysis approach to identify process parameters that should be examined during process characterization. Once these parameters are identified, a response surface study can be run to help understand the relationship between critical inputs and outputs.

Novel chemometric strategy based on the application of artificial neural networks to crossed mixture design for the improvement of recombinant protein production in continuous culture.

The optimal blends of six compounds that should be present in culture media used in recombinant protein production were determined by means of artificial neural networks (ANN) coupled with crossed mixture experimental design. This combination constitutes a novel approach to develop a medium for cultivating genetically engineered mammalian cells. The compounds were collected in two mixtures of three elements each, and the experimental space was determined by a crossed mixture design.

Suspension-Vero cell cultures as a platform for viral vaccine production.

Since Vero cells are currently considered as an acceptable cell substrate to produce a wide range of viruses, we developed a virus production platform using Vero cells adapted to grow in suspension in serum-free media. After adapting anchorage-dependent Vero cells to grow as a free-cell suspension, vesicular stomatitis virus, herpes simplex virus 1 and polio virus 1 production rates were evaluated in batch cultures using spinner flasks and perfused cultures in a bioreactor. The achieved results constitute valuable information for the development of a low-cost high-productivity process using a suspension culture of Vero cells to produce viral vaccines.

Rational selection of an antibody probe to detect the heterogeneous collection of CHO-derived rhGM-CSF glycoforms.

Six anti-E. coli rhGM-CSF monoclonal antibodies were generated using hybridoma technology. One of these showed identical affinity for the CHO and E.

Temperature reduction in cultures of hGM-CSF-expressing CHO cells: effect on productivity and product quality.

We have demonstrated that temperature reduction from 37 to 33 degrees C in the culture of a CHO cell line producing recombinant human granulocyte macrophage colony stimulating factor (CHO-K1-hGM-CSF) leads to a reduced growth rate, increased cell viability, improved cellular productivity, and decreased cell metabolism. In the present study, CHO-K1-hGM-CSF cells were cultured in a biphasic mode: first, a 37 degrees C growth phase for achieving a high cell number, followed by a production phase where the culture temperature was shifted to 33 degrees C. The maximum cell density was not affected after temperature reduction while cell viability remained above 80% for a further 3.

N- and O-linked carbohydrates and glycosylation site occupancy in recombinant human granulocyte-macrophage colony-stimulating factor secreted by a Chinese hamster ovary cell line.

GM-CSF is one of several naturally occurring glycoproteins that regulate leukocyte production, migration and function. It has been produced in different cell types, with different properties that depend on the production process used. The purpose of this work was to characterize the recombinant human GM-CSF from an engineered Chinese hamster ovary cell line grown in suspension and as adherent culture for the identification of the glycosylation sites and the definition of the glycosidic moiety, including the degree of site occupancy.