Some 160 biopharmaceuticals have now gained medical approval and several hundred are in the pipeline. Most are protein-based, although two nucleic acid-based products are now on the US/European market. An increasing proportion of approvals are engineered in some way and advances in alternative production systems and delivery methods will also likely impact upon the approvals profile over the remainder of this decade.

There are major structural differences between plant and mammalian N-linked glycans, with those from plants being immunogenic in most laboratory mammals and eliciting glycan-specific IgE and IgG antibodies in humans, when delivered parenterally. However, because humans are constantly exposed to plant glycoproteins in the diet, glycosylated plant-made pharmaceuticals (PMPs) should be acceptable for topical and oral administration. To exploit fully the potential that plants offer for the production of therapeutic proteins for parenteral administration, it might be necessary to inhibit plant-specific post-translational modifications to obtain 'humanized' non-immunogenic N-glycans on PMPs. The benefits that could accrue are lower manufacturing costs, relative to mammalian cell culture, and a reduced risk of transmission of mammalian pathogens.

Graft polymerization using potassium diperiodatocuprate as initiator was found to be an effective and convenient method for grafting functional polymer of N,N-dimethylaminoethyl methacrylate (DMAEMA) onto superporous polyacrylamide gels, so-called cryogels (pAAm cryogels). It was possible to achieve grafting degrees up to 110% (w/w). The two-step graft polymerization i.e. first activation of the matrix followed by displacement of initiator solution with the monomer solution, decreased pronouncedly the soluble homopolymer formation. The efficiency of graft polymerization using a two-step technique increased up to 50% (w/w) at a monomer conversion of 10%, compared to 10% graft efficiency with 60-70% monomer conversion for one-step direct graft polymerization. The pAAm cryogels grafted in one-step and two-step procedures, respectively, behaved similarly when binding low-molecular weight ligand but showed very different behavior for sorption of a high-molecular-weight ligand, bovine serum albumin (BSA). The differences in behavior were rationalized assuming different structure of the graft polymer layers and tentacle-type BSA binding to the grafted polymer.

Potassium diperiodatocuprate-initiated graft polymerization was found to be an efficient and convenient method for grafting of acrylic acid (AAc) onto superporous polyacrylamide gels, so called cryogels (pAAm cryogels). It was possible to achieve grafting degrees as high as 70% with about 25% yield of grafted polymer with respect to the initial amount of monomer. The superporous structure of the cryogels promoted grafting by providing an ample surface of the gel for grafting, ensuring good mass transfer inside the gel sample and allowing to wash easily both homopolymer of AAc and insoluble by-products formed during the polymerization reaction. The grafted cryogels could be dried at 60 [deg]C and re-swollen with retaining their properties. The adsorption of water vapours by dried pAAm cryogels was marginally dependent on the degree of grafting. The swelling of AAc-grafted pAAm cryogel increased pronouncedly with increasing pH. The adsorption of low-molecular weight ligand, Cu(II), by AAc-grafted pAAm cryogels increased linearly with the degree of grafting, while binding of high-molecular weight ligand, lysozyme, increased linearly till the degree of grafting of about 40% followed by a sharp, nearly three-fold increase in lysozyme binding when the degree of grafting increased from 60 to 70%. The results indicate that a 'tentacle'-type binding of lysozyme to grafted polyAAc takes place after a certain degree of grafting has been reached.

Polymerase chain reaction (PCR) is an in vitro enzymatic, synthetic method used to amplify specific DNA sequences from organisms. Detection of target DNA using gene probes allows for absolute identification not only of specific organisms, but also of genetic material in recombinant organisms. A major challenge facing detection of DNA from field samples is that they are almost sure to contain impurities, especially impurities that inhibit amplification through PCR. For example, humic material even in quantities as small as 1 ng have been shown to inhibit PCR. DNA has been extracted from sewage/stool samples, food, sputum, water and sediment, and human DNA likewise is being extracted from sources as varied as forensic samples of blood, cigarette butts and human remains. However, multi-step, time consuming methods are required to isolate the DNA from the surrounding contamination. This research focuses on developing a method for rapid cleanup of DNA which combines extraction and purification of DNA while, at the same time, removing inhibitors from ‘dirty samples’ to produce purified, PCR-ready DNA. GeneReleaser(TM) produces PCR-ready DNA in a rapid 5-min protocol. We report here the rapid extraction/purification of plasmid DNA from recombinant Escherichia coli. GeneReleaser(TM), inhibitor and ~103 cfu recombinant E. coli containing a plasmid insert were added to PCR tubes, vortexed for 30 s and microwaved for 5 min. DNA was PCR-amplified and identified by gel electrophoresis. GeneReleaser(TM) (GR) resin was able to cleanup samples containing typical aerosol and water/soil contaminants (dust--60 [mu]g, pollen--100 [mu]g, soot--250 [mu]g, humic acid--75 ng). While these inhibitors were easily removed prior to PCR amplification, other complex inhibitors found in soil and food samples remain a major challenge and detection of DNA in these materials typically requires multi-step procedures that may take up to a day. The advantages of using GR are that it is rapid and inexpensive.

The preparation of high quality plasmid DNA is a necessary requirement for most molecular biology applications. We compared four different large plasmid preparation protocols, which were based on either a liquid-phase approach (Triton lysis) or purification of alkaline lysis bacterial extracts followed by supercoiled plasmid purification on affinity columns. Two host Escherichia coli strains, JM 109 and INV[alpha]F′, were used to grow the test plasmids for comparison of product plasmid DNA produced from the four different plasmid isolation methods. While the DNA grown in E. coli strain JM109, prepared by liquid-phase Triton lysis was appropriately restricted by 12 restriction enzymes, this was not the case for any of the JM109-grown DNA purified by any of the affinity column solid-phase approaches. In contrast to this, when the plasmid DNA was grown in E. coli strain INV[alpha]F′, most restriction enzymes cut DNA appropriately, irregardless of the plasmid preparation protocol used. It seems that an impurity commonly eluted with the DNA from all three of the solid-phase DNA columns had an equal effect on the above enzymes using the common host strain JM109, but not strain INV[alpha]F′.

Pharmaceutical-grade plasmid DNA for use in vaccines and gene therapy requires the development of reproducible and scaleable downstream processes. Shearing of chromosomal DNA at the commencement of the purification results in fragments that are difficult to separate from supercoiled plasmid DNA. Regulatory standards will probably require that the level of chromosomal DNA contamination is kept below 0.01 mg mg-1 plasmid DNA. This work reports the use of nitrocellulose membranes to decrease chromosomal DNA contamination in plasmid DNA preparations derived from a 450-l bioreactor. Clarified lysates, resuspended PEG precipitates and anion exchange chromatography elutes were filtered through nitrocellulose. In all the cases, chromosomal DNA was selectively retained by the membrane while most supercoiled plasmid DNA was recovered in the filtrate. Contamination levels dropped from over 27% to below 1% as measured by Southern analysis. Under ionic strength conditions equal to or above 1.5 M NaCl, a fraction of the contaminant RNA was also retained by the nitrocellulose membrane.