Characterisation of a harvesting step for monoclonal antibodies from mammalian cell culture

The focus of this research was defined by a poorly characterised filtration train employed to clarify culture broth containing monoclonal antibodies secreted by GS-NSO cells: the filtration train blinded unpredictably and the ability of the positively charged filters to adsorb DNA from process material was unknown. To direct the development of an assay to quantify the ability of depth filters to adsorb DNA, the molecular weight of DNA from a large-scale, fed-batch, mammalian cell culture vessel was evaluated as process material passed through the initial stages of the purification scheme. High molecular weight DNA was substantially cleared from the broth after passage through a disc stack centrifuge and the remaining low molecular weight DNA was largely unaffected by passage through a series of depth filters and a sterilising grade membrane. Removal of high molecular weight DNA was shown to be coupled with clarification of the process stream. The DNA from cell culture supernatant showed a pattern of internucleosomal cleavage of chromatin when fractionated by electrophoresis but the presence of both necrotic and apoptotic cells throughout the fermentation meant that the origin of the fragmented DNA could not be unequivocally determined. An intercalating fluorochrome, PicoGreen, was elected for development of a suitable DNA assay because of its ability to respond to low molecular weight DNA. It was assessed for its ability to determine the concentration of DNA in clarified mammalian cell culture broths containing pertinent monoclonal antibodies. Fluorescent signal suppression was ameliorated by sample dilution or by performing the assay above the pI of secreted IgG. The source of fluorescence in clarified culture broth was validated by incubation with RNase A and DNase I. At least 89.0 % of fluorescence was attributable to nucleic acid and pre-digestion with RNase A was shown to be a requirement for successful quantification of DNA in such samples. Application of the fluorescence based assay resulted in characterisation of the physical parameters governing adsorption of DNA by various positively charged depth filters and membranes in test solutions and the DNA adsorption profile of the manufacturing scale filtration train. Buffers that reduced or neutralised the depth filter or membrane charge, and those that impeded hydrophobic interactions were shown to affect their operational capacity, demonstrating that DNA was adsorbed by a combination of electrostatic and hydrophobic interactions. Production-scale centrifugation of harvest broth containing therapeutic protein resulted in the reduction of total DNA in the process stream from 79.8 μg m1-1 to 9.3 μg m1-1 whereas the concentration of DNA in the supernatant of pre-and post-filtration samples had only marginally reduced DNA content: from 6.3 to 6.0 μg m1-1 respectively. Hence the filtration train was shown to ineffective in DNA removal. Historically, blinding of the depth filters had been unpredictable with data such as numbers of viable cells, non-viable cells, product titre, or process shape (batch, fed-batch, or draw and fill) failing to inform on the durability of depth filters in the harvest step. To investigate this, key fouling contaminants were identified by challenging depth filters with the same mass of one of the following: viable healthy cells, cells that had died by the process of apoptosis, and cells that had died through the process of necrosis. The pressure increase across a Cuno Zeta Plus 10SP depth filter was 2.8 and 16.5 times more sensitive to debris from apoptotic and necrotic cells respectively, when compared to viable cells. The condition of DNA released into the culture broth was assessed. Necrotic cells released predominantly high molecular weight DNA in contrast to apoptotic cells which released chiefly low molecular weight DNA. The blinding of the filters was found to be largely unaffected by variations in the particle size distribution of material in, and viscosity of, solutions with which they were challenged. The exceptional response of the depth filters to necrotic cells may suggest the cause of previously noted unpredictable filter blinding whereby a number of necrotic cells have a more significant impact on the life of a depth filter than a similar number of viable or apoptotic cells. In a final set of experiments the pressure drop caused by non-viable necrotic culture broths which had been treated with DNase I or benzonase was found to be smaller when compared to untreated broths: the abilities of the enzyme treated cultures to foul the depth filter were reduced by 70.4% and 75.4% respectively indicating the importance of DNA in the blinding of the depth filter studied.

A greenhouse integrating desalination, water saving and rainwater harvesting for use in salt-affected inland regions

This study proposes a new type of greenhouse for water re-use and energy saving for agriculture in arid and semi-arid inland regions affected by groundwater salinity. It combines desalination using reverse osmosis (RO), re-use of saline concentrate rejected by RO for cooling, and rainwater harvesting. Experimental work was carried at GBPUAT, Pantnagar, India. Saline concentrate was fed to evaporative cooling pads of greenhouse and found to evaporate at similar rates as conventional freshwater. Two enhancements to the system are described: i) A jet pump, designed and tested to use pressurized reject stream to re-circulate cooling water and thus maintain uniform wetness in cooling pads, was found capable of multiplying flow of cooling water by a factor of 2.5 to 4 while lifting water to a head of 1.55 m; and ii) Use of solar power to drive ventilation fans of greenhouse, for which an electronic circuit has been produced that uses maximum power-point tracking to maximize energy efficiency. Re-use of RO rejected concentrate for cooling saves water (6 l d-1 m-2) of greenhouse floor area and the improved fan could reduce electricity consumption by a factor 8.

A potentially scalable method for the harvesting of hMSCs from microcarriers

The use of hMSCs for allogeneic therapies requiring lot sizes of billions of cells will necessitate large-scale culture techniques such as the expansion of cells on microcarriers in bioreactors. Whilst much research investigating hMSC culture on microcarriers has focused on growth, much less involves their harvesting for passaging or as a step towards cryopreservation and storage. A successful new harvesting method has recently been outlined for cells grown on SoloHill microcarriers in a 5L bioreactor [1]. Here, this new method is set out in detail, harvesting being defined as a two-step process involving cell 'detachment' from the microcarriers' surface followed by the 'separation' of the two entities. The new detachment method is based on theoretical concepts originally developed for secondary nucleation due to agitation. Based on this theory, it is suggested that a short period (here 7min) of intense agitation in the presence of a suitable enzyme should detach the cells from the relatively large microcarriers. In addition, once detached, the cells should not be damaged because they are smaller than the Kolmogorov microscale. Detachment was then successfully achieved for hMSCs from two different donors using microcarrier/cell suspensions up to 100mL in a spinner flask. In both cases, harvesting was completed by separating cells from microcarriers using a Steriflip® vacuum filter. The overall harvesting efficiency was >95% and after harvesting, the cells maintained all the attributes expected of hMSC cells. The underlying theoretical concepts suggest that the method is scalable and this aspect is discussed too. © 2014 The Authors.