Case study and application of process analytical technology (PAT) towards bioprocessing: II. Use of ultra-performance liquid chromatography (UPLC) for making real-time pooling decisions for process chromatography

Process analytical technology (PAT) has been gaining a lot of momentum in the biopharmaceutical community due to the potential for continuous real-time quality assurance resulting in improved operational control and compliance. Two of the key goals that have been outlined for PAT are "variability is managed by the process" and "product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions". Recently, we have been examining the feasibility of applying different analytical tools for designing PAT applications for bioprocessing. We have previously shown that a commercially available online high performance liquid chromatography (HPLC) system can be used for analysis that can facilitate real-time decisions for column pooling based on product quality attributes (Rathore et al., 2008). In this article we test the feasibility of using a commercially available ultra- performance liquid chromatography (UPLC) system for real-time pooling of process chromatography columns. It is demonstrated that the UPLC system offers a feasible approach and meets the requirements of a PAT application. While the application presented here is of a reversed phase assay, the approach and the hardware can be easily applied to other modes of liquid chromatography.     

Production of lactic acid using a new homofermentative Enterococcus faecalis isolate

Lactic acid is an intermediate-volume specialty chemical for a wide range of food and industrial applications such as pharmaceuticals, cosmetics and chemical syntheses. Although lactic acid production has been well documented, improved production parameters that lead to reduced production costs are always of interest in industrial developments. In this study, we describe the production of lactic acid at high concentration, yield and volumetric productivity utilizing a novel homofermentative, facultative anaerobe Enterococcus faecalis CBRD01. The highest concentration of 182 g lactic acid l⁻¹ was achieved after 38 h of fed-batch fermentation on glucose. The bacterial isolate utilized only 2–13% of carbon for its growth and energy metabolism, while 87–98% of carbon was converted to lactic acid at an overall volumetric productivity of 5 g l⁻¹ h⁻¹. At 13 h of fermentation, the volumetric productivity of lactate production reached 10.3 g l⁻¹ h⁻¹, which is the highest ever reported for microbial production of lactic acid. The lactic acid produced was of high purity as formation of other metabolites was less than 0.1%. The present investigation demonstrates a new opportunity for enhanced production of lactic acid with potential for reduced purification costs.     

Regulation of the mutually exclusive exons 8a and 8 in the CaV1.2 calcium channel transcript by polypyrimidine tract-binding protein

CaV1.2 calcium channels play roles in diverse cellular processes such as gene regulation, muscle contraction, and membrane excitation and are diversified in their activity through extensive alternative splicing of the CaV1.2 mRNA. The mutually exclusive exons 8a and 8 encode alternate forms of transmembrane segment 6 (IS6) in channel domain 1. The human genetic disorder Timothy syndrome is caused by mutations in either of these two CaV1.2 exons, resulting in disrupted Ca(2+) homeostasis and severe pleiotropic disease phenotypes. The tissue-specific pattern of exon 8/8a splicing leads to differences in symptoms between patients with exon 8 or 8a mutations. Elucidating the mechanisms controlling the exon 8/8a splicing choice will be important in understanding the spectrum of defects associated with the disease. We found that the polypyrimidine tract-binding protein (PTB) mediates a switch from exon 8 to 8a splicing. PTB and its neuronal homolog, nPTB, are widely studied splicing regulators controlling large sets of alternative exons. During neuronal development, PTB expression is down-regulated with a concurrent increase in nPTB expression. Exon 8a is largely repressed in embryonic mouse brain but is progressively induced during neuronal differentiation as PTB is depleted. This splicing repression is mediated by the direct binding of PTB to sequence elements upstream of exon 8a. The nPTB protein is a weaker repressor of exon 8a, resulting in a shift in exon choice when nPTB replaces PTB in cells. These results provide mechanistic understanding of how these two exons, important for human disease, are controlled.     

A Coarse Grained Model for a Lipid Membrane with Physiological Composition and Leaflet Asymmetry

The resemblance of lipid membrane models to physiological membranes determines how well molecular dynamics (MD) simulations imitate the dynamic behavior of cell membranes and membrane proteins. Physiological lipid membranes are composed of multiple types of phospholipids, and the leaflet compositions are generally asymmetric. Here we describe an approach for self-assembly of a Coarse-Grained (CG) membrane model with physiological composition and leaflet asymmetry using the MARTINI force field. An initial set-up of two boxes with different types of lipids according to the leaflet asymmetry of mammalian cell membranes stacked with 0.5 nm overlap, reliably resulted in the self-assembly of bilayer membranes with leaflet asymmetry resembling that of physiological mammalian cell membranes. Self-assembly in the presence of a fragment of the plasma membrane protein syntaxin 1A led to spontaneous specific positioning of phosphatidylionositol(4,5)bisphosphate at a positively charged stretch of syntaxin consistent with experimental data. An analogous approach choosing an initial set-up with two concentric shells filled with different lipid types results in successful assembly of a spherical vesicle with asymmetric leaflet composition. Self-assembly of the vesicle in the presence of the synaptic vesicle protein synaptobrevin 2 revealed the correct position of the synaptobrevin transmembrane domain. This is the first CG MD method to form a membrane with physiological lipid composition as well as leaflet asymmetry by self-assembly and will enable unbiased studies of the incorporation and dynamics of membrane proteins in more realistic CG membrane models.     

The influence of substituents (R) at N atom of thiophene-2-carbaldehyde thiosemicarbazones {(C4H3S)HCN-N(H)-C(S)NHR} on bonding, nuclearity and H-bonded networks of copper(I) complexes

The nuclearity, bonding and H-bonded networks of copper(I) halide complexes with thiophene-2-carbaldehyde thiosemicarbazones {(C₄H₃S)HC²N³-N(H)-C¹(S)N¹HR} are influenced by R substituents at N¹ atom. Thiophene-2-carbaldehyde-N¹-methyl thiosemicarbazone (HttscMe) or thiophene-2-carbaldehyde-N¹-ethyl thiosemicarbazone (HttscEt) have yielded halogen-bridged dinuclear complexes, [Cu₂(μ-X)₂(η¹-S-Htsc)₂(Ph₃P)₂] (Htsc, X: HttscMe, I, 1; Br, 2; Cl, 3; HttscEt, I, 4; Br, 5; Cl, 6), while thiophene-2-carbaldehyde-N¹-phenyl thiosemicarbazone (HttscPh) has yielded mononuclear complexes, [CuX(η¹-S-HttscPh)₂] (X, I, 7a; Br 8; Cl, 9) and a sulfur bridged dinuclear complex, [Cu₂(μ-S-HttscPh)₂(η¹-S-HttscPh)₂I₂] 7b co-existing with 7a in the same unit cell. These results are in contrast to S-bridged dimers [Cu₂(μ-S-Httsc)₂(η¹-Br)₂(Ph₃P)₂] · 2H₂O and [Cu₂(μ-S-Httsc)₂(η¹-Cl)₂(Ph₃P)₂] · 2CH₃CN obtained for R = H and X = Cl, Br (Httsc = thiophene-2-carbaldehyde thiosemicarbazone) as reported earlier. The intermolecular CH_Ph?π interaction in 1-3 (2.797 Å, 1; 3.264 Å, 2; 3.257 Å, 3) have formed linear polymers, whereas the CH_Ph?X and N³?HCH interactions in 4-6 (2.791, 2.69 Å, 5; 2.776, 2.745 Å, 6, respectively) have led to the formation of H-bonded 2D polymer. The PhN¹H?π, interactions (2.547 Å, 8, 2.599 Å, 9) have formed H-bonded dimers only. The Cu?Cu separations are 3.221-3.404 Å (1-6).     

Trans-cellular introduction of HIV-1 protein Nef induces pathogenic response in Caenorhabditis elegans

Background

Caenorhabditis elegans has emerged as a very powerful model for studying the host pathogen interactions. Despite the absence of a naturally occurring viral infection for C. elegans, the model is now being exploited experimentally to study the basic aspects of virus-host interplay. The data generated from recent studies suggests that the virus that infects mammalian cells does infect, replicate and accumulate in C. elegans.

Methodology/Principal Findings

We took advantage of the easy-to-achieve protein introduction in C. elegans and employing the methodology, we administered HIV-1 protein Nef into live worms. Nef is known to be an important protein for exacerbating HIV-1 pathogenesis in host by enhancing viral replication. The deletion of nef from the viral genome has been reported to inhibit its replication in the host, thereby leading to delayed pathogenesis. Our studies, employing Nef introduction into C. elegans, led to creation of an in-vivo model that allowed us to study, whether or not, the protein induces effect in the whole organism. We observed a marked lipodystrophy, effect on neuromuscular function, impaired fertility and reduced longevity in the worms exposed to Nef. The observed effects resemble to those observed in Nef transgenic mice and most interestingly the effects also relate to some of the pathogenic aspects exhibited by human AIDS patients.

Conclusions/Significance

Our studies underline the importance of this in vivo model for studying the interactions of Nef with host proteins, which could further be used for identifying possible inhibitors of such interactions.     

Biodegradation of glyceryl trinitrate by Penicillium corylophilum Dierckx.

Penicillium corylophilum Dierckx, isolated from a contaminated water wet, double-base propellant, was able to completely degrade glyceryl trinitrate (GTN) in a buffered medium (pH 7.0) containing glucose and ammonium nitrate. In the presence of 12 mg of initial fungal inoculum, GTN (48.5 to 61.6 mumol) was quantitatively transformed in a stepwise process to glyceryl dinitrate (GDN) and glyceryl mononitrate (GMN) within 48 h followed by a decrease in the GDN content with a concomitant increase in the GMN level. GDN was totally transformed to GMN within 168 h, and the complete degradation of GMN was achieved within 336 h. The presence of glucose and ammonium nitrate in the growth medium was essential for completion of the degradation of GTN and its metabolites. Complete degradation of GTN by a fungal culture has not been previously reported in the literature.     

The messenger RNA sequences in human fibroblast cells induced with poly rI.rC to produce interferon

Induction of human fibroblast cells with poly rI.rC induces interferon mRNA which can be translated into interferon precursor in wheat germ cell free system or in Xenopus oocytes into biologically active interferon. The extent of gene expression in the poly rI.rC induced cells was compared to that of the uninduced cells by hybridization of the mRNA to complementary DNA. Homologous template driven hybridization of cDNA revealed the presence of two clearly defined transitions in the total poly A RNA from the induced cells; abundant class and a scarce class comprising approximately 37,000 diverse species of RNA. Heterologus hybridization of the cDNA with total uninduced mRNA showed that the majority of the mRNA sequences are the same in both the induced and uninduced cells. The results of the hybridization using cDNA prepared to the fraction enriched for interferon mRNA, however, showed that about 4% of the sequences present in the interferon enriched fraction are not present in the uninduced cells. These differences may result from the poly rI.rC induced alterations in gene expression.     

Influence of inoculum size of Aspergillus parasiticus spores on aflatoxin production.

The influence of the inoculum size on growth and aflatoxin production was examined in Aspergillus parasiticus (NRRL 3145) by using a synthetic medium. The reduction in the number of spores by 4 to 5 log cycles either by serial dilution or by gamma irradiation caused a two fold increase in the toxin production. The decrease in the inoculum size induced a lag in growth of the culture, though the final yield of the mycelium over the 28-day experimental period was the same. The maximal accumulation of aflatoxin was observed on day 14 of incubation. A transition from the biphasic to monophasic pattern in aflatoxin production could be correlated with the size of the inoculum. The enhanced toxin production from dilute inocula was similar to that obtained with the surviving fraction of the spores after gamma irradiation (0 to 150 krads).