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Background The quality of biologicals, including biosimilars, is subject to change as a result of manufacturing process modifications following initial authorization. It is important that such product changes have no adverse impact on product efficacy or safety, including immunogenicity. Objectives The aim of this study was to investigate the number and types of manufacturing changes for originator mAbs (the reference for the comparability exercise to confirm biosimilarity) according to European Public Assessment Report (EPAR) documentation and to ascertain the level of risk these changes might impart. The extensive body of evidence contained in the EPAR documents can help support the EMA during the EC marketing authorization approval process for biosimilars, since it provides a broad base of scientific experience. Research designs and methods For EPAR-listed mAbs, details of all changes listed chronologically in the EPAR were evaluated and described. Based on these descriptions the manufacturing changes can be categorized by risk status (low, moderate or high). Results Entries for 29 mAbs with publicly available EPAR reports were reviewed. These contained details of 404 manufacturing changes authorized by the European Medicines Agency (EMA): 22 were categorized as high risk, 286 as moderate risk and 96 as low risk manufacturing changes. A limitation of this analysis is that it only summarizes publicly available data from EPAR documents. Conclusions Manufacturing change data indicate that the EMA has significant experience of process changes for originator mAbs, and the impact they may have on the efficacy and safety of biologicals. This experience will be useful in biosimilar product development to ensure adherence to sound scientific principles. Compared with the established manufacturing process for a reference product, the production of biosimilars will usually be different. Consequently, in addition to a comprehensive comparative functional and physicochemical characterization analysis, clinical data is required to confirm mAb biosimilarity.

Genes encoding the tail proteins of the temperate phage 16-3 of the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti 41 have been identified. First, a new host range gene, designated hII, was localized by using missense mutations. The corresponding protein was shown to be identical to the 85-kDa tail protein by determining its N-terminal sequence. Electron microscopic analysis showed that phage 16-3 possesses an icosahedral head and a long, noncontractile tail characteristic of the Siphoviridae. By using a lysogenic S. meliloti 41 strain, mutants with insertions in the putative tail region of the genome were constructed and virion morphology was examined after induction of the lytic cycle. Insertions in ORF017, ORF018a, ORF020, ORF021, the previously described h gene, and hII resulted in uninfectious head particles lacking tail structures, suggesting that the majority of the genes in this region are essential for tail formation. By using different bacterial mutants, it was also shown that not only the RkpM and RkpY proteins but also the RkpZ protein of the host takes part in the formation of the phage receptor. Results for the host range phage mutants and the receptor mutant bacteria suggest that the HII tail protein interacts with the capsular polysaccharide of the host and that the tail protein encoded by the original h gene recognizes a proteinaceous receptor.

Understanding the molecular mechanisms underlying bone development is a fundamental and fascinating problem in developmental biology, with significant medical implications. Here, we have identified the expression patterns for 36 genes that were characteristic or dominant in the consecutive cell differentiation zones (mesenchyme, precartilage, cartilage) of the tip section of the developing velvet antler of red deer Cervus elaphus. Two major functional groups of these genes clearly outlined: six genes linked to high metabolic demand and other five to tumor biology. Our study demonstrates the advantages of the antler as a source of mesenchymal markers, for distinguishing precartilage and cartilage by different gene expression patterns and for identifying genes involved in the robust bone development, a striking feature of the growing antler. Putative roles for "antler" genes that encode alpha-tropomyosine (tpm1), transgelin (tagln), annexin 2 (anxa2), phosphatidylethanolamine-binding protein (pebp) and apolipoprotein D (apoD) in intense but still controlled tissue proliferation are discussed.

A high-throughput device has been constructed which allows parallel electroelution of separated SDS-protein bands directly from intact unsectioned polyacrylamide gel slabs as well as single electroelution of certain protein spots into a 384-well standard flat-bottom multiwell plate. The prototype provides complete, quick elution for proteomics from 1-D or from 2-D gels without gel sectioning. Since the elution chamber matrix requires no assembly, sample handling can be easily carried out by existing robotic workstations. The current design is a good candidate for automation of spot elution since there are no moving liquid containing components in the apparatus. Eight SDS-proteins were eluted in test runs and an average 70% sample recovery was achieved by re-electrophoresis of the electro-eluates.

In Europe, a long history of cooperation over transboundary rivers--most notably the Rhine and Danube rivers--exists. To help foster cooperation and communication vis-à-vis transboundary ground water, the United Nations Economic Commission for Europe (UNECE), as part of its ground water program, conducted a survey on transboundary aquifers in Europe. The survey produced 25 responses from 37 countries and identified 89 transboundary aquifers. Respondents reported on the degree of ground water use within their own boundaries, transboundary aspects (agreements, joint commissions, etc.) of ground water, and transboundary aquifers themselves. The inventory proved useful, but a number of problems were identified: different map scales and symbols, difficulty in identifying transboundary aquifers, inconsistent labeling of aquifers, and data discrepancies. The UNECE ground water program also drafted guidelines for monitoring and assessment of transboundary ground water. These guidelines are not legally binding but have been adopted by 25 countries, deal mainly with monitoring and assessment, and are being implemented through a number of pilot projects. Other organizations-the United Nations Scientific, Educational and Cultural Organization, the Food and Agriculture Organization, the International Association of Hydrogeologists, and the European Union--are all supporting the investigation of transboundary aquifers in an effort to facilitate data sharing and coordinated management of these valuable resources.

Cohesive ends of 16-3, a temperate phage of Rhizobium meliloti 41, have been identified as 10-base-long, 3'-protruding complementary G/C-rich sequences. terS and terL encode the two subunits of 16-3 terminase. Significant homologies were detected among the terminase subunits of phage 16-3 and other phages from various ecosystems.

In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), moving boundaries I-IV (Table 1) form. They migrate isotachophoretically at displacement rates that increase in the order of I to II to III. Moving boundaries IV and V comprising Pyronin-SDS as a leading constituent are retarded at high gel concentrations in comparison with the isotachophoretically migrating species. Since analytes depending on their net mobilities stack within any of those moving boundaries, previous R(f) values and Ferguson plots may have to be revised.

Separated protein bands are sequentially electrophoresed into low melting agarose plugs distributed in an apparatus of original design along the surface of a plastic drum. The rotation of the drum is synchronized to migration of electrophoretic bands to receive each band individually. Agarose plugs are dissolved enzymatically for transfer into the mass spectrometer. One microL of the agarose solution containing 1 pmol of each of three lithium and natrium salts of dodecyl sulfate (Li-Na-DS)-proteins were applied to matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) without any prepurification. It yields a signal indistinguishable from that obtained in the absence of agarose.

A discontinuous Tris-Cl/acetate (OAc) buffer system, unprecedently containing OAc as the trailing constituent, and operative in polyacrylamide gel electrophoresis (PAGE) at low polyacrylamide concentration (T = 4.8%) is described in the paper. The characteristics of the electrophoretic system are illustrated by the resolution of fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS)-labeled malto-oligosaccharides and dextran homopolymers. In this buffer system, the resolving phase is constituted by Tris-OAc behind a moving boundary formed between the leading chloride ion of Tris-HCl gel buffer and the trailing OAc ion provided by a catholyte of NH(4)OAc. In contrast with the results obtained with Tris-CI/glycinate buffer commonly used in electrophoresis, or with Tris-CI/borate, the best resolution of the glucose oligomers containing 1-4 glucose units in Tris-OAc, pH 8.8, ionic strength of 0.08, was obtained at 4.8% polyacrylamide concentration, using 0.5 M NH(4)OAc, pH 9.5 as the catholyte. Under those conditions, the ANTS-glucose oligomers were separated with mobilities decreasing from glucose to maltohexaose. The linear Ferguson plots (log relative mobility, R(f), vs.%T) of the glucose oligomers show that the surface net charge of those oligomers is inversely related to their sizes, given by the slopes, K(R), of the plots. The molecular weight of the oligomers is directly but nonlinearly related to K(R). The novel electrophoretic system illustrated here for separation of short ANTS-saccharides can be potentially applied to the resolution of other biomolecules such as rapidly migrating DNA, peptides or proteins.

16-3 is a temperate phage of the symbiotic nitrogen-fixing bacterium Rhizobium meliloti 41. Its prophage state and immunity against superinfection by homoimmune phages are governed by a complex set of controls: the immC and immX repressor systems and the avirT element are all located in well-separated, distinct regions which span 25 kb on the bacteriophage chromosome. The anatomy and function of the immC region are well documented; however, fewer analyses have addressed the immX and avirT regions. We focused in this paper on the immX region and dissected it into two major parts: X(U/L) and X(V). The X(U/L) part (0.6 kb) contained two overlapping cistrons, X(U) and X(L), coding for proteins pXU and pXL, respectively. Inactivation of either gene inactivated the repressor function of the immX region. Loss-of-function mutants of X(U) and X(L) complemented each other in trans in double lysogens. The X(V) part (1 kb) contained a target for X(U/L) repressor action. Mutations at three sites in X(V) led to various degree of ImmX insensitivity in a hierarchic manner. Two sites (X(V1) and X(V3)) exhibited the inverted-repeat structures characteristic of many repressor binding sites. However, X(V1) could also be folded into a transcription terminator. Of the two immunity regions of 16-3, immX seems to be unique both in its complex genetic anatomy and in its sequence. To date, no DNA or peptide sequence homologous to that of ImmX has been found in the data banks. In contrast, immC shares properties of a number of immunity systems commonly found in temperate phages.

Prokaryotic repressor-operator systems provide exemplars for the sequence-specific interactions between DNA and protein. The crucial atomic contacts of the two macromolecules are attained in a compact, geometrically defined structure of the DNA-protein complex. The pitch of the DNA interface seems an especially sensitive part of this architecture because changes in its length introduce new spacing and rotational relations in one step. We discovered a natural system that may serve as a model for investigating this problem: the repressor of the 16-3 phage of Rhizobium meliloti (helix-turn-helix class protein) possesses inherent ability to accommodate to various DNA twistings. It binds the cognate operators, which are 5'-ACAA-4 bp-TTGT-3' (O(L)) and 5'-ACAA-6 bp-TTGT-3' (O(R)) and thus differ 2 bp in length, and consequently the two half-sites will be rotated with respect to each other by 72 degrees in the idealized B-DNA (64 degrees by dinucleotide steps calculations). Furthermore, a synthetic intermediate (DNA sequence) 5'-ACAA-5 bp-TTGT-3' (O5) also binds specifically the repressor. The natural operators and bound repressors can form higher order DNA-protein complexes and perform efficient repression, whereas the synthetic operator-repressor complex cannot do either. The natural operators are bent when complexed with the repressor, whereas the O5 operator does not show bending in electrophoretic mobility assay. Possible structures of the complexes are discussed.

An electroelution apparatus prototype of a new design was constructed. In that design, the electric field passes vertically through the protein band located on a horizontal (PhastSystem) minigel polymerized on a net of Gel-Fix (Serva). A simple, home-made apparatus allows for electroelution of protein bands at the level of a few picomoles and their identification, after concentration, by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The technique is applicable to one-dimensional (1-D) or two-dimensional (2-D) gels of any size, but has been exemplified only by application to 1-D minigels to demonstrate the lower limits of protein load of the method. When in the course of further development of the prototype it will be combined with a modification to two dimensions of the electroelution mechanism under computer control of the high-performance gel electrophoresis apparatus (formerly of LabIntelligence), the new design appears uniquely qualified for an automated spot elution from 2-D gels under avoidance of gel sectioning.

In this paper we present an HPLC method developed for quick activity and specificity analysis of serine proteinases. The method applies a carefully designed peptide library in which the individual components differ only at the potential cleavage site for enzymes. The library has seven members representing seven different cleavage sites and it offers substrates for both trypsin and chymotrypsin-like enzymes. The individual peptide substrates compete for the proteinase during the enzymatic reaction. The reaction is monitored by RP-HPLC separation of the components. We describe the systematic design of the competitive peptide substrate library and the test of the system with eight different serine proteinases. The specificity profiles of the investigated enzymes as determined by the new method were essentially identical to the ones reported in the literature, verifying the ability of the system to characterize substrate specificity. The tests also demonstrated that the system could detect even subtle specificity differences of two isoforms of an enzyme. In addition to recording qualitative specificity profiles, data provided by the system can be analyzed quantitatively, yielding specificity constant values. This method can be a useful tool for quick analysis of uncharacterized gene products as well as new forms of enzymes generated by protein engineering., (Copyright 2001 Academic Press.)

It has been known since 1990 that DNA curvature can be recognized on transverse pore gradient gels by an intersection of "Ferguson curves" with those of DNA size standards. The miniaturized PhastSystem polyacrylamide gels allow one to detect DNA curvature effortlessly and fast and at great economy of sample relative to alternative methods of electrophoresis. Using the transverse gradient gel electrophoresis method, it was found that the 660 bp length subfragment of the matrix attachment region (MAR) sequence of the chicken lysosyme gene migrates as a fragment of 800-900 bp length. When subjected to digestion with the restriction enzyme HaeIII, the fragment gives rise to two species of 248 and 412 bp length, respectively. The Ferguson curves of both species intersect with those of DNA size standards, indicating that both exhibit curvature. Only the curvature of the 412 bp fragment conforms to prediction. Ethidium bromide abolishes the effect of curvature on the fragment, reducing its apparent size from 900 to 660, the value obtained by agarose gel electrophoresis.

Two peptides, SGCI and SGTI, that inhibited chymotrypsin and trypsin, respectively, were isolated from the haemolymph of Schistocerca gregaria. Their primary structures were found to be identical with SGP-2 and SGP-1, two of a series of peptides isolated from ovaries of the same species (A. Hamdaoui et al., FEBS Lett. 422 (1998) 74-78). All these peptides are composed of 35-36 amino acid residues and contain three homologous disulfide bridges. The residues imparting specificity to SGCI and SGTI were identified as Leu-30 and Arg-29, respectively. The peptides were synthesised by solid-phase peptide synthesis, and the synthetic ones displayed the same inhibition as the natural forms: SGCI is a strong inhibitor of chymotrypsin (K(i) = 6.2 x 10(-12) M), and SGTI is a rather weak inhibitor of trypsin (K(i) = 2.1 x 10(-7) M). The replacement of P(1) then P(1)' residues of SGCI with trypsin-specific residues increased affinity towards trypsin 3600- and 1100-fold, respectively, thus SGCI was converted to a strong trypsin inhibitor (K(i) = 5.0 x 10(-12) M) that retained some inhibitory affinity towards chymotrypsin (K(i) = 3.5 x 10(-8) M). The documented role of both P(1) and P(1)' highlights the importance of S(1)'P(1)' interactions in enzyme-inhibitor complexes.

différences! [editorial] [editorial]onomic way of identifying and assigning nematodes to taxons, which had already been determined either by comparative sequence analysis of nuclear rDNA internal transcribed spacer (ITS) region or by other methods of molecular or conventional taxonomy, is provided. Molecular identification of entomopathogenic nematodes (EPN) can be upgraded by basing it on PhastSystem polyacrylamide gel electrophoresis (PAGE) analysis of restriction fragment length polymorphism (RFLP) patterns of polymerase chain reaction (PCR)-amplified DNA derived from single nematodes of Steinernema or Heterorhabditis spp. Although analysis from single worms has previously been made on agarose gel, the resolution on PhastSystem PAGE gel is much higher. The DNA sequences selected for analysis were those constituting the internal transcribed spacer region between the 18S and 26S rDNA genes within the rRNA operon. RFLP analysis was carried out by gel electrophoresis on the PhastSystem (Pharmacia) as detailed elsewhere (Triga et al., Electrophoresis 1999, 20, 1272-1277. The downscaling from conventional agarose to PhastSystem gels resulted in pattern of DNA fragments differing from those obtained with agarose gel electrophoresis under conventional conditions by increasing the number of detected fragments. The approach supported previous species identifications and was able to identify several unclassified isolates, such as those from Hungary and Ireland, and provides a method for identification of previously unclassified strains. We confirmed that Heterorhabditis "Irish Type", represented by two strains of different geographical origin, comprise a species different from H. megidis. We also confirmed that strain IS5 belongs to the species H. indicus rather than to H. bacteriophora, as had been suggested previously.

The DNA sequences constituting the internal transcribed spacer region, located between 18S and 26S rDNA genes within the rRNA operon, derived from single nematodes of two genera (Steinernema and Heterorhabditis) were amplified by polymerase chain reaction (PCR) and subjected to digestion by four restriction enzymes. The digests were analyzed by restriction fragment length polymorphism (RFLP) gel electrophoresis on the PhastSystem, using 7.5%T, 5%C(Bis) polyacrylamide. The downscaling from conventional agarose to PhastSystem gels permitted the analysis to be done on individual nematodes, rather than on mixed samples with average properties. The analysis time was reduced so as to allow for the electrophoretic separation on 200 samples/workday. The resulting patterns of DNA fragments differed from those obtained by agarose gel electrophoresis under conventional conditions by an increased number of detected fragments. The PhastSystem gel analysis provides the basis for taxonomical revisions.

Recombinant vesicle-associated membrane protein (rVAMP), implicated as a participant in membrane exocytosis and fusion (a "SNARE protein"), was subjected to gel electrophoresis in the miniaturized gels of the PhastSystem (Pharmacia) containing the nondenaturing, nonionic detergent beta-octylglucoside (OG), followed by immunodetection of the protein. Three major components of nondenatured rVAMP are detected by Western blotting both in 0.5% OG and in the absence of detergent. Their separation increases with increasing gel concentration above 7%T. Ferguson plot analysis indicates that the three species of VAMP are size isomers (i.e., they differ in size but share a common surface net charge density), the common point of intersection of the plots (mu-point) being a measure of their common free mobility. By the criteria of size and free mobility (related to surface net charge), VAMP components I, II and III in 0.5% OG-containing buffer are indistinguishable from components II, III and IV, respectively, observed in the absence of the detergent. The feasibility of immunodetection of nondenatured rVAMP on gels containing nondenaturing detergents opens up the possibility of gaining biochemical information regarding nondenatured SNARE protein complexes and SNARE proteins linked to membrane fragments.

The Escherichia coli resident mobile element IS30 has pronounced target specificity. Upon transposition, the element frequently inserts exactly into the same position of a preferred target sequence. Insertion sites in phages, plasmids and in the genome of E. coli are characterized by an exceptionally long palindromic consensus sequence that provides strong specificity for IS30 insertions, despite a relatively high level of degeneracy. This 24-bp-long region alone determines the attractiveness of the target DNA and the exact position of IS30 insertion. The divergence of a target site from the consensus and the occurrence of 'non-permitted' bases in certain positions influence the target activity. Differences in attractiveness are emphasized if two targets are present in the same replicon, as was demonstrated by quantitative analysis. In a system of competitive targets, the oligonucleotide sequence representing the consensus of genomic IS30 insertion sites proved to be the most efficient target. Having compared the known insertion sites, we suppose that IS30-like target specificity, which may represent an alternative strategy in target selection among mobile elements, is characteristic of the insertion sequences IS3, IS6 and IS21, too.

The application of pore gradient gels prefabricated for the PhastSystem (Pharmacia) to transverse pore gradient gel electrophoresis is demonstrated. It has the twofold advantage of (i) horizontal positioning, avoiding gel stretching during the preparation of these gels and resulting pore size irreproducibility experienced with vertically applied pore gradient gels, which necessitate an orthogonal transfer of spacers, and (ii) miniaturized gel dimensions, which allow a small sample load and a short duration of electrophoresis and staining.

The amino acid sequence of the 27 kDa protein responsible for the haemolytic activity of Bacillus thuringiensis subsp. israelensis toxin has been analysed by secondary structure prediction, helical wheel/net diagrams and molecular mechanics calculations. We found that segment 116-126 presumably forms a strongly amphiphilic alpha-helix. This is supported by the findings that the synthesized segment 116-126 (a) has a significant alpha-helical content in water, and (b) displays an in vitro haemolytic activity comparable to that of bee venom peptide melittin. As segment 116-126 is present in the haemolyzing, but not present in the non-haemolyzing proteins from B. thuringiensis toxins, we suggest that this segment is responsible for the lytic potential of the B. thuringiensis subsp. israelensis protein.

Factors influencing the operation of a vertical bioreactor segmented with perforated plates supporting immobilized yeast cells were studied. It was found that the most important factors are the length-diameter (L/D) ratio of the reactor and the dilution rate. It was supposed that the optimal L/D ratio is about 1. The operation of the reactor was more favourable at a low liquid phase-solid phase ratio. The spatial distribution of the biocatalyst had only a slight, if any effect. The periodically changed direction of feed flow has no improving effect on the fermentation process.