Upstream processes in antibody production: evaluation of critical parameters

The demand for monoclonal antibody for therapeutic and diagnostic applications is rising constantly which puts up a need to bring down the cost of its production. In this context it becomes a prerequisite to improve the efficiency of the existing processes used for monoclonal antibody production. This review describes various upstream processes used for monoclonal antibody production and evaluates critical parameters and efforts which are being made to enhance the efficiency of the process. The upstream technology has tremendously been upgraded from host cells used for manufacturing to bioreactors type and capacity. The host cells used range from microbial, mammalian to plant cells with mammalian cells dominating the scenario. Disposable bioreactors are being promoted for small scale production due to easy adaptation to process validation and flexibility, though they are limited by the scale of production. In this respect Wave bioreactors for suspension culture have been introduced recently. A novel bioreactor for immobilized cells is described which permits an economical and easy alternative to hollow fiber bioreactor at lab scale production. Modification of the cellular machinery to alter their metabolic characteristics has further added to robustness of cells and perks up cell specific productivity. The process parameters including feeding strategies and environmental parameters are being improved and efforts to validate them to get reproducible results are becoming a trend. Online monitoring of the process and product characterization is increasingly gaining importance. In total the advancement of upstream processes have led to the increase in volumetric productivity by 100-fold over last decade and make the monoclonal antibody production more economical and realistic option for therapeutic applications.     

<Emphasis Type="SmallCaps">L</Emphasis>(+)-Lactic Acid Production Using <Emphasis Type="Italic">Lactobacillus Casei</Emphasis> in Solid-State Fermentation

Lactobacillus casei was grown at 37 °C on sugarcane bagasse (5 g) soaked with cassava starch hydrolysate (final moistening volume 34 ml) containing 3 g reducing sugar in a solid-state condition. The maximum yield of l-lactic acid after various process optimisations was 2.9 g/5 g initial substrate corresponding to 97% conversion of sugar to lactic acid with initial substrate moisture of 72%.     

Reactive oxygen species mediate Endothelin-1-induced activation of ERK1/2, PKB, and Pyk2 signaling, as well as protein synthesis, in vascular smooth muscle cells

Reactive oxygen species (ROS) have been shown to mediate the effects of several growth factors and vasoactive peptides, such as epidermal growth factor, platelet-derived growth factor, and angiotensin II (AII). Endothelin-1 (ET-1) is a vasoactive peptide which also exhibits mitogenic activity in vascular smooth muscle cells (VSMCs), and is believed to contribute to the pathogenesis of vascular abnormalities such as atherosclerosis, hypertension, and restenosis after angioplasty. However, a possible role for ROS generation in mediating the ET-1 response on extracellular signal-regulated kinases 1 and 2 (ERK1/2), protein kinase B (PKB), and protein tyrosine kinase 2 (Pyk2), key components of the growth-promoting and proliferative signaling pathways, has not been examined in detail. Our aim was to investigate the involvement of ROS in ET-1-mediated activation of ERK1/2, PKB, and Pyk2 in A-10 VSMCs. ET-1 stimulated ERK1/2, PKB, and Pyk2 phosphorylation in a dose- and time-dependent manner. Pretreatment of A-10 VSMCs with diphenyleneiodonium (DPI), an inhibitor of reduced nicotinamide adenine dinucleotide phosphate oxidase, attenuated ET-1-enhanced ERK1/2, PKB, and Pyk2 phosphorylation. In addition, in parallel with an inhibitory effect on the above signaling components, DPI also blocked ET-1-induced protein synthesis. ET-1 was also found to increase ROS production, which was suppressed by DPI treatment. N-Acetylcysteine, a ROS scavenger, exhibited a response similar to that of DPI and inhibited ET-1-stimulated ERK1/2, PKB, and Pyk2 phosphorylation. These results demonstrate that ROS are critical mediators of ET-1-induced signaling events linked to growth-promoting proliferative and hypertrophic pathways in VSMCs.     

Enhanced arsenic accumulation in engineered bacterial cells expressing ArsR

The metalloregulatory protein ArsR, which offers high affinity and selectivity toward arsenite, was overexpressed in Escherichia coli in an attempt to increase the bioaccumulation of arsenic. Overproduction of ArsR resulted in elevated levels of arsenite bioaccumulation but also a severe reduction in cell growth. Incorporation of an elastin-like polypeptide as the fusion partner to ArsR (ELP153AR) improved cell growth by twofold without compromising the ability to accumulate arsenite. Resting cells overexpressing ELP153AR accumulated 5- and 60-fold-higher levels of arsenate and arsenite than control cells without ArsR overexpression. Conversely, no significant improvement in Cd(2+) or Zn(2+) accumulation was observed, validating the specificity of ArsR. The high affinity of ArsR allowed 100% removal of 50 ppb of arsenite from contaminated water with these engineered cells, providing a technology useful to comply with the newly approved U.S. Environmental Protection Agency limit of 10 ppb. These results open up the possibility of using cells overexpressing ArsR as an inexpensive, high-affinity ligand for arsenic removal from contaminated drinking and ground water.     

Peroxynitrite resistance of sarco/endoplasmic reticulum Ca2+ pump in pig coronary artery endothelium and smooth muscle

We examined the effects of peroxynitrite pre-treatment on sarco/endoplasmic reticulum Ca(2+) (SERCA) pump in pig coronary artery smooth muscle and endothelium. In saponin-permeabilized cells, smooth muscle showed much greater rates of the SERCA Ca(2+) pump-dependent (45)Ca(2+) uptake/mg protein than did the endothelial cells. Peroxynitrite treatment of cells inhibited the SERCA pump more severely in smooth muscle cells than in endothelial cells. To determine implications of this observation, we next examined the effect of the SERCA pump inhibitor cyclopiazonic acid (CPA) on intracellular Ca(2+) concentration of intact cultured cells. CPA produced cytosolic Ca(2+) transients in cultured endothelial and smooth muscle cells. Pre-treatment with peroxynitrite (200 microM) inhibited the Ca(2+) transients in the smooth muscle but not in the endothelial cells. CPA contracts de-endothelialized artery rings and relaxes precontracted arteries with intact endothelium. Peroxynitrite (250 microM) pre-treatment inhibited contraction in the de-endothelialized artery rings, but not the endothelium-dependent relaxation. Thus, endothelial cells appear to be more resistant than smooth muscle to the effects of peroxynitrite at the levels of SERCA pump activity, CPA-induced Ca(2+) transients in cultured cells, and the effects of CPA on contractility. The greater resistance of endothelium to peroxynitrite may play a protective role in pathological conditions such as ischemia-reperfusion when excess free radicals are produced.     

Effects of peptide molecular mass and PEG chain length on the vasoreactivity of VIP and PACAP(1-38) in pegylated phospholipid micelles

Bioactive properties of certain amphipathic peptides are amplified when self-associated with sterically stabilized micelles (SSM) composed of polyethylene glycol (PEG)-conjugated phospholipids. The purpose of this study was to determine the effects of amphipathic peptide molecular mass and PEG chain length on vasoreactivity evoked by vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide, and pituitary adenylate cyclase-activating peptide(1-38) (PACAP(1-38)), a 38-amino acid neuropeptide, associated with PEGylated phospholipid micelles in vivo. Both peptides were incubated for 2 h with SSM composed of PEG with molecular mass of 2000 or 5000 grafted onto distearoyl-phosphatidylethanolamine (DSPE-PEG2000 or DSPE-PEG5000) before use. We found that regardless of peptide molecular mass, PEG chain length had no significant effects on peptide-SSM interactions. Using intravital microscopy, VIP associated with DSPE-PEG5000 SSM or DSPE-PEG2000 SSM incubated at 25 degrees C evoked similar vasodilation in the intact hamster cheek pouch microcirculation. Likewise, PACAP(1-38)-induced vasodilation was PEG chain length-independent. However, SSM-associated PACAP(1-38) evoked significantly smaller vasodilation than that evoked by SSM-associated VIP (P < 0.05) at 25 degrees C. When the incubation temperature was increased to 37 degrees C, SSM-associated PACAP(1-38)-induced vasodilation was now similar to that of SSM-associated VIP. This response was associated with a corresponding increase in alpha-helix content of both peptides in the presence of phospholipids. Collectively, these data indicate that for a larger amphipathic peptide, such as PACAP(1-38), greater kinetic energy or longer incubation period is required to optimize peptide-SSM interactions and amplify peptide bioactivity in vivo.     

Yeast and mammalian alpha-glucosidase inhibitory constituents from Himalayan rhubarb Rheum emodi Wall.ex Meisson

The methanolic extract of rhizome of Himalayan rhubarb Rheum emodi displayed mild yeast as well as mammalian intestinal alpha-glucosidase inhibitory activity. However, further fractionation of active extract led to the isolation of several potent molecules in excellent yields, displaying varying degrees of inhibition on two test models of alpha-glucosidase. Rhapontigenin, desoxyrhapontigenin, chrysophanol-8-O-beta-d-glucopyranoside, torachrysone-8-O-beta-d-glucopyranoside displayed potent yeast alpha-glucosidase inhibition. However chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin and torachrysone-8-O-beta-d-glucopyranoside displayed potent to moderate mammalian alpha-glucosidase inhibitory activity. Other compounds displayed mild activity on both the tests. Except desoxyrhapontigenin and rhapontigenin that increased Vmax, other compounds including crude extract decreased the Vmax significantly (p<0.02) in yeast alpha-glucosidase test. Further kinetic analysis on mammalian alpha-glucosidase inhibition showed that chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin and torachrysone-8-O-beta-d-glucopyranoside may be classified as mixed-noncompetitive inhibitors. However, desoxyrhapontigenin and rhapontigenin may be classified as modulators of enzyme activity. Presence and position of glycoside moiety in compounds appear important for better inhibition of mammalian alpha-glucosidase. This is the first report assigning particularly, mammalian intestinal alpha-glucosidase inhibitory activity to these compounds. Chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin, desoxyrhapontigenin and rhapontigenin have been isolated in substantial yields from R. emodi for the first time. Therefore, these compounds may have value in the treatment and prevention of hyperglycemia associated diabetes mellitus.     

Identification of a potent and rapidly reversible inhibitor of the 20S-proteasome

Synthesis and in vitro characterization of novel, lactam boronic acid based, selective, and rapidly reversible inhibitor 14 of the 20S-proteasome is presented.