Automated alkaline lysis for industrial scale cGMP production of pharmaceutical grade plasmid-DNA

Plasmid DNA for biopharmaceutical applications is mainly produced in E. coli cells. The first and most crucial step for recovering the plasmid is the cell lysis. Governed by the physico-chemical properties of the polynucleotide, alkaline lysis has been the lysis-method of choice. This chemical disintegration technique was initially developed for the lab scale and non-pharmaceutical applications. A continuous, fully automated and closed system combining alkaline lysis, neutralization and clarification in one gentle and generic operation was developed. This system consists of a three units. One unit controls mixing and contact time during the alkaline treatment, another one controls the neutralization and the concurrent formation of flocs and a third one the separation of flocs and pDNA containing lysate. Based on optimization experiments the selected process parameters resulted in yields up to 100% and homogeneities comparable to that obtained by gentle manual lysis. The process does not need enzymes and it is scalable and routinely used for cGMP-production of pharmaceutical grade plasmid DNA from 200 L fermentations.     

Evidence for multiple sites of ferricyanide reduction in chloroplasts.

Various sites of ferricyanide reduction were studied in spinach chloroplasts. It was found that in the presence of dibromothymoquinone a fraction of ferricyanide reduction was dibromothymoquinone sensitive, implying that ferricyanide can be reduced by photosystem I as well as photosystem II. To separate ferricyanide reduction sites in photosystem II, orthophenanthroline and dichlorophenyl dimethylurea inhibitions were compared at various pHs. It was noted that at low pH ferricyanide reduction was not completely inhibited by orothophenanthroline. At high pH's, however, inhibition of ferricyanide reduction by orthophenanthroline was complete. It was found that varying concentration of orthophenanthroline at a constant pH showed different degrees of inhibition. In the study of ferricyanide reduction by photosystem II various treatments affecting plastocyanin were performed. It was found that Tween-20 or KCN treatments which inactivated plastocyanin did not completely inactivate ferricyanide reduction. These data support the conclusion that ferricyanide accepts electrons both before and after plastoquinone in photosystem II.     

Automated analysis of rat breast tumors. Comparison of four methods

Chemically-induced malignant rat breast tumors pose diagnostic dilemmas since the majority are well-differentiated, noninvasive papillary lesions that are barely distinguishable from benign papillary lesions. This study compared several automated modalities to see which best separated benign from malignant breast tumors. Thirty-three carcinogen-induced rat breast tumors (13 adenomas, 10 papillary carcinomas and 10 invasive carcinomas) were evaluated by static (image) cytometry (ICM) of integrated optical density, by flow cytometry (FCM) and by two automated morphometric protocols, contextual analysis and single-gland analysis. DNA ploidy analysis, by either ICM or FCM, did not discriminate between the benign and malignant tumors. Contextual analysis correctly identified 11 of 13 benign and 17 of 20 malignant lesions (P less than .01). Single-gland analysis correctly identified all 13 benign and 17 of 20 malignant lesions (P less than .01). No method distinguished invasive from noninvasive carcinomas. The data suggest that architectural features are more important than nuclear features in differentiating benign from malignant rat breast tumors.     

Non-destructive methods for demonstrating chemical changes in the rhizosphere II. Application of methods

Chemical changes in the rhizosphere of soil-grown plants are demonstrated by non-destructive techniques based on colour reactions. The following examples are given: FeIII reduction in the rhizosphere of a Hakea species, MnIV reduction in the rhizosphere of chikpea, complexation of Al in the rhizosphere of Norway spruce, and the activity of acid phosphatase in the rhizosphere of maize.     

Properties of placental alkaline phosphatase. II. Interactions of fast- and slow-migrating components

The relationship between the rapidly (component I) and slowly (components II) migrating components of placental alkaline phosphatase has been studied. Storage of components II resulted in conversion into component I with a parallel increase of activity. The conversion rate increased with temperature. The fastest of the slow-moving components (component II) was less stable than the very slow-migrating ones (components II). Component II was not identifiable after 25 days at 37 C, while some of the II components were still present after a period of 1 year. No conversion of component I into II was observed. Data from this study suggest that the apparent thermostability of placental alkaline phosphatase results partly from the increase in activity after the conversion of components II into component I.This work was supported in part by U.S. Public Health Service Grant HD02552. K.H. is a Career Scientist of the Health Research Council of the City of New York (I-513).     

Selectivity for maltose and maltodextrins of maltoporin, a pore-forming protein of E. coli outer membrane

Homogenous maltoporin (lamB protein), an Escherichia coli outer membrane spanning protein, was incorporated in phospholipid planar bilayers. It generates aqueous channels distinct from those formed by the non-specific porin (OmpF) or by phosphoporin (phoE protein). The single conductance, 150 pS in 1 M NaCl, is much smaller than that of the porins. The channels, which are poorly selective for cations and voltage independent, are specifically inhibited by maltose and maltodextrins. This inhibition, observed in the absence of maltose binding protein, demonstrates that the selectivity of maltoporin for maltose and maltodextrins is an intrinsic property of the protein.     

Automated docking of ligands to antibodies: methods and applications

Many approaches to studying protein-ligand interactions by computational docking are currently available. Given the structures of a protein and a ligand, the ultimate goal of all docking methods is to predict the structure of the resulting complex. This requires a suitable representation of molecular structures and properties, search algorithms to efficiently scan the configuration space for favorable interaction geometries, and accurate scoring functions to evaluate and rank the generated orientations. For many of the available methods, tests on experimentally known antibody-antigen or antibody-hapten complexes have appeared in the literature. In addition, some of them have been used in predictive studies on antibody-ligand interactions to provide structural insights where adequate experimental information is missing. The AutoDock program is presented as example of a method for flexibly docking ligands to antibodies. Applying parameters of the second-generation AMBER force field, three antibody-hapten complexes (AN02, DB3, NC6.8) are used as new test cases to analyze the ability of the method to reproduce experimental findings. The X-ray structures could be reconstituted and the corresponding solutions were ranked with best energy score in all cases. Docking to the free instead of the complexed NC6.8 structure indicated the limits of the rigid protein treatment, although fairly good guesses about the location of the binding site and the contact residues could still be obtained if conformational flexibility was allowed at least in the ligand.     

Class II recombinant phosphoribosyl diphosphate synthase from spinach. Phosphate independence and diphosphoryl donor specificity

A recombinant form of spinach (Spinacia oleracea) phosphoribosyl diphosphate (PRPP) synthase isozyme 3 resembling the presumed mature enzyme has been synthesized in an Escherichia coli strain in which the endogenous PRPP synthase gene was deleted, and has been purified to near homogeneity. Contrary to other PRPP synthases the activity of spinach PRPP synthase isozyme 3 is independent of P(i), and the enzyme is inhibited by ribonucleoside diphosphates in a purely competitive manner, which indicates a lack of allosteric inhibition by these compounds. In addition spinach PRPP synthase isozyme 3 shows an unusual low specificity toward diphosphoryl donors by accepting dATP, GTP, CTP, and UTP in addition to ATP. The kinetic mechanism of the enzyme is an ordered steady state Bi Bi mechanism with K(ATP) and K(Rib-5-P) values of 170 and 110 micrometer, respectively, and a V(max) value of 13.1 micromol (min x mg of protein)(-1). The enzyme has an absolute requirement for magnesium ions, and maximal activity is obtained at 40 degrees C at pH 7.6.     

Adrenodoxin reductase and adrenodoxin. Mechanisms of reduction of ferricyanide and cytochrome c.

Adrenodoxin reductase, the flavoprotein moiety of the adrenal cortex mitochondrial steroid hydroxylating system, participates in adrenodoxin-dependent cytochrome c and adrenodoxin-independent ferricyanide reduction, with NADPH as electron donor for both of these 1-electron reductions. For ferricyanide reduction, adrenodoxin reductase cycles between oxidized and 2-electron-reduced forms, reoxidation proceeding via the neutral flavin (FAD) semiquinone form (Fig. 9). Addition of adrenodoxin has no effect upon the kinetic parameters of flavoprotein-catalyzed ferricyanide reduction. For cytochrome c reduction, the adrenodoxin reductase-adrenodoxin 1:1 complex has been shown to be the catalytically active species (Lambeth, J. D., McCaslin, D. R., and Kamin, H. (1976) J. Biol. Chem. 251, 7545-7550). Present studies, using stopped flow techniques, have shown that the 2-electron-reduced form of the complex (produced by reaction with 1 eq of NADPH) reacts rapidly with 1 eq of cytochrome c (k approximately or equal to 4.6 s-1), but only slowly with a second cytochrome c (k = 0.1 to 0.3 s-1). However, when a second NADPH is included, two more equivalents of cytochrome are reduced rapidly. Thus, the adrenodoxin reductase-adrenodoxin complex appears to cycle between 1- and 3-electron reduced states, via an intermediate 2-electron-containing form produced by reoxidation by cytochrome (Fig. 10). For ferricyanide reduction by adrenodoxin reductase, the fully reduced and semiquinone forms of flavin each transfer 1 electron at oxidation-reduction potentials which differ by approximately 130 mV. However, adrenodoxin in a complex with adrenodoxin reductase allows electrons of constant potential to be delivered from flavin to cytochrome c via the iron sulfur center...     

Immune response to levan. II. T independence of suppression of cross-reactive idiotypes by anti-idiotype antibodies.

Pretreatment of BALB/c mice with antisera to a cross-reactive idiotype (E109IdX) expressed on many anti-bacterial levan (BL) and anti-inulin (Inu) antibodies leads to a prolonged suppression in production of IdX-bearing molecules in response to BL immunization. There is a comparable suppression in numbers of plaque-forming cells secreting IdX-bearing anti-BL and anti-Inu molecules. Furthermore, spleen cells from anti-E109IdX pretreated mice are unable to transfer to irradiated recipients the ability to produce IdX-bearing anti-BL and anti-Inu antibodies. These results indicate that the suppressive effect is at the precursor level and not simply a clearance of antibodies bearing the IdX. Suppression of IdX production can be achieved by pretreating nu/nu BALB/c mice with anti-E109IdX antibodies. Furthermore, spleen cells from pretreated mice do not inhibit the capacity of spleen cells from normal mice transferred to irradiated recipients to produce E109IdX in response to BL. This indicates that the suppression of IdX production in the anti-BL system is T independent and probably represents direct inhibition of precursors by anti-IdX.     

Automated methods for accurate determination of the critical velocity of packed bed chromatography

Knowing the critical velocity (ucrit) of a chromatography column is an important part of process development as it allows the optimization of chromatographic flow conditions. The conventional flow step method for determining ucrit is prone to error as it depends heavily on human judgment. In this study, two automated methods for determining ucrit have been developed: the automatic flow step (AFS) method and the automatic pressure step (APS) method. In the AFS method, the column pressure drop is monitored upon application of automated incremental increases in flow velocity, whereas in the APS method the flow velocity is monitored upon application of automated incremental increases in pressure drop. The APS method emerged as the one with the higher levels of accuracy, efficiency and ease of application having the greater potential to assist defining the best operational parameters of a chromatography column.     

Sensitivity of various visualization methods for peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry

Summary Various chromogen protocols for visualizing peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry were compared with respect to their sensitivity. They were tested on tissue sections of human skeletal muscle and in an antigen spot test using antibodies against slow skeletal muscle myosin. The chromogens included 3-amino-9-ethylcarbazole (AEC), 3, 3-diaminobenzidine (DAB),p-phenylenediamine-pyrocatechol (PPD-PC) and 4-chloro-1-naphthol (CN) in peroxidase histochemistry, and 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium salt (BCIP-NBT), BCIP-tetra nitro blue tetrazolium salt (TNBT) and various combinations of substituted naphthol phosphate-diazonium salt in alkaline phosphatase histochemistry. DAB, CN, and PPD-PC were also employed with imidazole and DAB in addition to Co²⁺ and Ni²⁺ ions. The results indicate that DAB-imidazole and DAB-Co²⁺ and Ni²⁺ ions are the most sensitive chromogen protocols for visualizing peroxidase activity. Although no large differences were found between the various chromogen protocols for visualizing alkaline phosphatase activity, the protocol BCIP-TNBT is especially recommended. Furthermore, the various chromogen protocols were evaluated as to stability of chromogen solutions and final precipitates, background staining, localization properties, and enhancement of enzyme activity.