TRPV1 is a non-selective cation channel gated by noxious heat, vanilloids and extracellular protons, and act as an important signal integrator in sensory nociceptors. Because of its integrative signaling properties in response to inflammatory stimuli, TRPV1 antagonists are predicted to inhibit the sensation of ongoing or burning pain that is reported by patients suffering from chronic pain, therefore offering an unprecedented advantage in selectively inhibiting painful signaling from where it is initiated. In this chapter, we firstly summarize the physiological and pathological roles of TRPV1 and then describe the pharmacology of TRPV1 agonists and antagonists. Finally, we give an update and the status on TRPV1 therapies that have progressed into clinical trials.

A fast and convenient method for baculovirus quantification in microplate format has been developed. The cell diameter was chosen as the measuring parameter, since insect cells are enlarged upon infection, and swelling is proportional to the amount of virus particles in the solution (Liu, 2004). Insect cells were seeded into a 96-well microplate, infected with serial dilutions of baculoviruses, and incubated at 27°C. After 24 h, the cell diameter average in each well was determined in an automated image-based screening system. Dose response curves resulting from infections of viruses with known and unknown titres, respectively, were compared, and the titres of the unknown viruses calculated from the midpoint shift. This approach enabled simultaneous quantification of five viruses in one 96-well plate and could be carried out in the presence of cell growth media.

To avoid the time consuming, labor intensive seed‐train expansion and to improve production reliability and consistency, portions of bulk cryopreserved cells from the same cultivation can be utilized as inocula or alternatively may be used to undertake transient transfections for large‐scale bioreactor production. In this study, the conditions for large‐scale freezing in cryobags were optimized utilizing a design of experiment approach. We showed that relatively high density of 30–40 × 106 cells/mL and relatively low Me2SO concentrations of 5–6% in the freezing media are optimal to freeze HEK293‐EBNA and CHO‐S cells in a controlled manner in order to achieve high viable cell recovery and growth post‐thawing. The immediate transfer of freshly thawed cells into culture medium resulted in better cell growth compared to cells that were centrifuged in order to remove Me2SO. This was the case as long as the residual Me2SO did not exceed 0.2–0.3%. The best time to perform transient 25 kDa polyethylenimine‐mediated transfection of pCEP4‐EGFP plasmid into freshly thawed, one‐step inoculated cells is after 72–96 h in culture. At this time point, the numbers of EGFP‐positive cells in the freshly thawed culture mimic perfectly that of cells grown continuously. Finally, our data showed that it is possible to freeze transiently polyethyleneimine‐transfected HEK293‐EBNA cells and maintain growth rate and expression of recombinant protein following thawing. The optimal time point for freezing cells was 4 h after transfection. Biotechnol. Bioeng. 2008;100: 911–922. © 2008 Wiley Periodicals, Inc.

We have developed a generic transient transfection process at 100 L scale, using HEK293-EBNA cells and PEI as the transfection reagent for the production of recombinant IgG. The process, including large-scale plasmid preparation, expression at bioreactor scale, capture, purification and, if necessary, endotoxin removal allows reproducible production of more than 0.5 g IgG for in vitro and in vivo studies. We compared the performance of two HEK cell lines, investigated the effect of conditioned medium, optimized the DNA:PEI ratio and implemented a feed strategy to prolong the culture time to increase product yield. The transient transfection protocol developed enables a closed process from seeding culture to protein capture. The challenge of performing a medium exchange before transfection at large scale is solved by applying a continuous centrifugation step between the seeding bioreactor and the production bioreactor. After 7–8 days the harvest and capture is performed in a one-step operation using a Streamline expanded bed chromatography system. Following a polishing step the purified antibody is transferred to the final formulation buffer. The method has shown to be reproducible at 10, 50, and 100 L scale expressing between 5 and 8 mg L −1 IgG.

Bioreactor culture of adherent cells offers clear advantages for scale-up of cells for membrane preparations. However, harvesting cells from microcarriers is time-consuming. In addition, enzymatic treatment may lead to loss of ligand binding and functional activity. To address this, we investigated scalable procedures to streamline cell harvesting and membrane preparation avoiding the use of enzymatic treatment. The concept is based on wash of cell-loaded micro-carriers in the fermentor, harvest and freezing of the cells directly without enzymatic treatment. Starting from the thawed carrier-cell material we compared enzymatically treated with non treated membrane preparations with respect to yield of functionally active receptors.

Transient transfection of mammalian cells with episomal vectors is a very useful method for producing high levels of recombinant proteins. Transient systems remove the need for the laborious and time-consuming process of creating stable cell lines. Here, we describe the optimisation and evaluation of a high-throughput transient expression system in HEK293-EBNA cells. The process was developed for the expression of 10 constructs simultaneously in deep-well plates and subsequent purification using 96-well plate affinity chromatography. This enabled multiple combinations of different constructs, vectors, and expression conditions to be studied in parallel.

Conditioned medium (CM) taken from a serum-free culture of Trichoplusia ni (BTI-Tn-5B1-4, High Five) cells on days 2 and 3, shortened the lagphase and increased the maximum cell density when added to T. ni cultures with low-inoculum cell density. Gel filtration fractions of CM, eluting at around 45kDa, stimulated cell proliferation even better than CM. A protein in the gel filtration fraction was identified by N-terminal amino acid sequencing as a proteinase, related to a snake venom metalloproteinase. Casein zymography showed, multiple metalloproteinase bands between 48 and 25kDa, as well as precursor forms above 48kDa. Metalloproteinase bands below the main band at 48kDa were autocatalytic degradation products. Metalloproteinase activity was the sole factor responsible for the growth stimulating effect of CM as shown by using the specific metalloproteinase inhibitor dl-thiorphan. Metalloproteinases have recently been shown to release growth factors from sequestering extracellular proteins. We propose that the metalloproteinase is involved in autocrine regulation of T. ni proliferation in serum-free media. In addition, a gel filtration fraction of CM, eluting at about 10kDa, inhibited cell growth. Apart from a lysozyme precursor protein and a cyclophilin-like protein, a kazal-type proteinase inhibitor could be identified in this fraction.

The G-protein coupled melanocortin 4 receptor (MC4r) plays an important role in the energy metabolism. We overexpressed the MC4r in CHO cells and performed characterisation studies on the cell membranes to determine functional stability and ligand binding properties of the receptor. The affinity for the ligands [Nle4, d-Phe7]-αMSH and MTII was lost below pH 6 but could be restored by returning to physiological pH. Increasing NaCl concentration up to 1 M had little influence on the binding of either ligand. At neutral pH, physiological salt concentration and 4 °C the ligand affinity of the receptor was stable for up to 6 days. These findings will facilitate design of purification methods for the receptor.

beta-Secretase is one of the prime targets for therapeutic intervention of Alzheimer's disease. For the development of a secretase inhibitor a steady supply of large quantities of a homogeneous and active recombinant beta-secretase is a prerequisite. Therefore various culture modes were investigated using HEK-293 cells stably transfected with soluble recombinant beta-secretase. The coupling of the Fc part of human IgG1 to the ectodomain of beta-secretase (residues 1-460) allowed a fast purification of the protein with rProtA expanded bed chromatography. Batch cultures of 5 to 50 L working volume run for 7 days showed reproducible cell growth and product yields of 3 mg/L purified protein. A 20 L perfusion culture was operated for 21 days, reaching a cell density of 30 x 10(6) cells/mL at a dilution rate of 2/d. The total product yield of the perfusion culture was 1.4 g of purified protein. The effect of different perfusion rates on cell growth, protein yield, and quality was investigated and compared to the results obtained in batch cultures. Protein quality was consistent as analyzed on 1D SDS-PAGE, and the final product contained both the mature and the pro form of beta-secretase. Although the cell specific protein expression was slightly reduced in perfusion culture, a substantial increase in specific activity of over 75% was achieved. Some of the increase in activity can be explained by an increase in the percentage of the mature form of the recombinant protein.

Two model G-protein coupled membrane receptors (GPCRs), aserotonin (5HT) and a metabotropic glutamate (mGlu) receptor, stablyexpressed in CHO cells were used to characterize cultureconditions for maximum receptor expression and functionalactivity in membrane preparations. Expression levels of the5HT receptor were affected by the growth phase of the cellculture. Maximum receptor density, as measured by ligandbinding per mg membrane protein, was observed when cells wereharvested in late exponential growth phase. Expression couldbe increased further by addition of 10 mM sodium butyrate andincubation at 31 °C for 24 hours prior to cellharvest. In contrast, functional activity as determined byagonist-stimulated GTPγS binding was independent of the growthrate. For both receptors, butyrate treatment at decreasedtemperature negatively affected functional activity. The mGlureceptor membranes lost functional activity considerably whenthe cells were cultured in an agitated system either onmicrocarriers or as aggregates in suspension. Functionalactivity could be restored and further improved compared to acontrol grown in T-flasks when the cell culture was incubatedat 31 °C for 48 hours following a complete mediumexchange and omission of sodium butyrate.

The aim of the study was to develop a scalable and robust generic process for production of recombinant cytosolic proteins in High Five cells using the baculo virus expression vector system (BEVS). The increasing demand for recombinant proteins in drug discovery has led to requirement for a generic protocol for protein supply. The process itself should fit into an ordinary weekly working scheme. The challenge was that the activity of many proteins could only be analysed when highly purified. Additionally, many proteins were extremely sensitive to protease degradation and could only be stored after purification. The influence of multiplicity of infection (MOI) and different growth media on protein expression was investigated. Time of infection and time of harvest was thoroughly evaluated with respect to high protein quality.

The speed and success in process development and optimisation can be enhanced by application of a small-scale screening system. A new screening reactor system for cultivation of animal cells has been characterised regarding oxygen transfer and working parameters. Based on this characterisation an optimised configuration was evaluated with Tricopulsia ni (High Five cells) prior and after baculo virus infection.

Monoclonal IgA was produced under serum free conditions by a murine hybridoma cell line (ZAC3) in a hollow fibre, a continuous stirred tank and a fluidized bed reactor. Differences in the antibody adsorption to DEAE chromatography matrices, an essential step in downstream processing, were related to the production systems. Chromatography on hydroxyapatite was used to separate monomeric, dimeric and polymeric IgA. This method was successfully applied to IgA produced by all three reactor configurations. The binding of dimeric and polymeric IgA to antigen was tested by ELISA. Using 2-dimensional SDS-PAGE analysis, dimeric IgA from the three sources was shown to be identical with respect to isoelectric points of alpha, kappa and J-chain but showed marked differences in purity. Finally the efficiency of the three bioprocesses was assessed by comparing the product yields after purification. This included the reactor specific production rates, media and time requirements and time consumption for the production of 1 g purified dimeric IgA.

A novel downstream process is reported for preparative scale production of dimeric Immunoglobulin A (IgA), a protein with potential pharmaceutical applications. Time-consuming size exclusion chromatography was replaced by ceramic hydroxyapatite chromatography (CHT). A clear baseline separation of the dimeric IgA from the polymeric forms using a step-gradient-step protein elution program is described. Application of this method for IgA analysis during production is suggested.

Anti- V.cholerae monoclonal IgA antibodies have been produced using a hybridoma cell line (Zac3) grown in RPMI medium containing 10% Foetal calf serum (FCS) and a completely defined protein- free medium. The latter has been optimised with respect to amino acid, pluronic, glucose and glutamine content to give batch growth, total IgA concentration, IgA productivity and dimeric IgA fraction which is even higher than that obtained during growth on the complex medium. The hybridoma has been grown in batch and continuous modes in a range of bioreactor configurations and the quantity and quality of the IgA produced determined. The results show that high cell density systems (hollow fibre reactors) produce the highest concentrations and productivities. However, the high concentration can result in the formation of high levels of polymeric forms which creates difficulties for the downstream processing. The bioreactor configuration had an important effect on IgA productivity, ratio of mono-, di- and poly- meric forms as well as antigen binding activity.