Continuous optical in-line glucose monitoring and control in CHO cultures contributes to enhanced metabolic efficiency while maintaining darbepoetin alfa product quality.

Great efforts are directed towards improving productivity, consistency and quality of biopharmaceutical processes and products. One particular area is the development of new sensors for continuous monitoring of critical bioprocess parameters by using online or in-line monitoring systems. Recently, we developed a glucose biosensor applicable in single-use, in-line and long-term glucose monitoring in mammalian cell bioreactors. Now, we integrated this sensor in an automated glucose monitoring and feeding system capable of maintaining stable glucose levels, even at very low concentrations. We compared this fed-batch feedback system at both low (< 1 mM) and high (40 mM) glucose levels with traditional batch culture methods, focusing on glycosylation and glycation of the recombinant protein darbepoetin alfa (DPO) produced by a CHO cell line. We evaluated cell growth, metabolite and product concentration under different glucose feeding strategies and show that continuous feeding, even at low glucose levels, has no harmful effects on DPO quantity and quality. We conclude that our system is capable of tight glucose level control throughout extended bioprocesses and has the potential to improve performance where constant maintenance of glucose levels is critical.

Investigating the dynamics of recombinant protein secretion from a microalgal host.

Production of recombinant proteins with microalgae represents an alternative platform over plant- or bacterial-based expression systems for certain target proteins. Secretion of recombinant proteins allows accumulation of the target product physically separate from the valuable algal biomass. To date, there has been little investigation into the dynamics of recombinant protein secretion from microalgal hosts-the culture parameters that encourage secreted product accumulation and stability, while encouraging biomass production. In this work, the efficiency of recombinant protein production was optimized by adjusting cultivation parameters for a strain of Chlamydomonas reinhardtii previously engineered to secrete a functional recombinant Lolium perenne ice binding protein (LpIBP), which has applications as a frozen food texturing and cryopreservation additive, into its culture medium. Three media and several cultivation styles were investigated for effects on secreted LpIBP titres and culture growth. A combination of acetate and carbon dioxide feeding with illumination resulted in the highest overall biomass and recombinant protein titres up to 10mgL(-1) in the culture medium. Pure photoautotrophic production was possible using two media types, with recombinant protein accumulation in all cultivations correlating to culture cell density. Two different cultivation systems were used for scale-up to 10L cultivations, one of which produced yields of secreted recombinant protein up to 12mgL(-1) within six cultivation days. Functional ice recrystallization inhibition (IRI) of the LpIBP from total concentrated extracellular protein extracts was demonstrated in a sucrose solution used as a simplified ice cream model. IRI lasted up to 7 days, demonstrating the potential of secreted products from microalgae for use as food additives.

Robust and selective nano cavities for protein separation: an interpenetrating polymer network modified hierarchically protein imprinted hydrogel.

In the present work, we report a novel method for the reinforcement of hierarchically structured molecularly imprinted polymer (MIP) for the separation of human serum albumin (HSA) and immunoglobulin G (IgG) proteins under pressure driven flow conditions. The template proteins (HSA or IgG) were first physically adsorbed at their isoelectric point on the surface of wide pore silica particles. Thereafter, the pore system was filled with a monomer solution and polymerized to form a lightly crosslinked polyacrylamide network covering the protein template. In order to enhance the rigidity of the hydrogels, different type of crosslinkers such as ethylene glycol dimethacrylate (EGDMA), 3,4-dihydroxyphenethylamine (DA) and methylene-bis-acrylamide (MBA), at least partially interpenetrated into the initially made acrylamide base hydrogel leading to formation of an interpenetrating polymer network (IPN). Then the silica matrix was removed to leave highly porous and reinforced MIP. TGA together with FT-IR and TEM analysis supported the interpenetration of the secondary crosslinkers in the initially formed polymer matrix. The compression property of the modified hydrogels as a function of degree of swelling was in the following order: DA>EGDMA>MBA-IPN-modified hydrogel. Batch binding assay verified the capability of the IPN modified MIPs to capture the target proteins. Moreover, solid phase extraction, HPLC and SDS-PAGE revealed that the EGDMA modified MIP could selectively capture and separate the proteins from human serum or fermentation broth.

Flexible automation of cell culture and tissue engineering tasks.

Until now, the predominant use cases of industrial robots have been routine handling tasks in the automotive industry. In biotechnology and tissue engineering, in contrast, only very few tasks have been automated with robots. New developments in robot platform and robot sensor technology, however, make it possible to automate plants that largely depend on human interaction with the production process, e.g., for material and cell culture fluid handling, transportation, operation of equipment, and maintenance. In this paper we present a robot system that lends itself to automating routine tasks in biotechnology but also has the potential to automate other production facilities that are similar in process structure. After motivating the design goals, we describe the system and its operation, illustrate sample runs, and give an assessment of the advantages. We conclude this paper by giving an outlook on possible further developments.

Serum-free production of a chimeric E-selectin-IgG protein from 1 to 100 l scale: Repeated batch cultivation versus continuous spin filter perfusion.

On inflamed endothelium the cell surface protein E-selectin isexpressed which supports the initial process of attachment -capturing and rolling of leukocytes. A recombinant CHO cell linesecreting a soluble E-selectin-IgG chimera was cultivated competitively under serum free conditions in three different bioreactor systems: a 1 l Super-Spinner, a 2 l stirred tank bioreactor equipped with a spinfilter, and a 100 l stirred tankbioreactor. In the smallest system 25.4 mg E-selectin-IgG wereproduced in 62 days using a repeated batch process whileachieving a maximal viable cell density of 3.7 x 10(6) cells ml(-1). Using continuous perfusion mode a total amount of35.2 mg were produced with a maximal viable cell density of1.65 x 10(7) cells ml(-1) in the 2 l bioreactor within 29 days. Large scale cultivation in a 100 l stirred tankbioreactor yielded 105.6 mg in three batches with a maximal viable cell density of 9.7 x 10(5) cells ml(-1) within 15 days. After removal of the cells by continuous centrifugation and a depth filter clearance step, the supernatants were concentrated via ultra filtration. Purificationwas performed by affinity chromatography with rProtein A. Integrity of the E-selectin-IgG protein was checked with SDS PAGE. Its activity was verified in a cellular adhesion assay performed with HL-60 cells and a recombinant CHO cell line expressing membrane-anchored E-selectin constitutively, and E-selectin expressing HUVECs, respectively. Soluble E-selectin-IgG was used to block adhesion to these cell layerscompetitively. A concentation of 18.8 and 37.5 mug ml(-1)was sufficient to reduce the amount of adhering HL-60 cells to 50% on CHO and HUVEC layers, respectively.

New technologies for automated cell counting based on optical image analysis ;The Cellscreen'.

A prototype of a newly developed apparatus for measuring cell growth characteristics of suspension cells in micro titre plates over a period of time was examined. Fully automated non-invasive cell counts in small volume cultivation vessels, e.g. 96 well plates, were performed with the Cellscreen system by Innovatis AG, Germany. The system automatically generates microscopic images of suspension cells which had sedimented on the base of the well plate. The total cell number and cell geometry was analysed without staining or sampling using the Cedex image recognition technology. Thus, time course studies of cell growth with the identical culture became possible. Basic parameters like the measurement range, the minimum number of images which were required for statistically reliable results, as well as the influence of the measurement itself and the effect of evaporation in 96 well plates on cell proliferation were determined. A comparison with standard methods including the influence of the cultured volume per well (25 mul to 200 mul) on cell growth was performed. Furthermore, the toxic substances ammonia, lactate and butyrate were used to show that the Cellscreen system is able to detect even the slightest changes in the specific growth rate.