Stability Study Through Water Activity Measurements for Dispensed Powdered Raw Materials.

BACKGROUND: Holding times for raw materials are relevant since they enable us to understand the allowable time that a raw material can be kept under ideal storage conditions before the start of the manufacturing process without its quality attributes being affected. The quantification of water activity can be used as an indicator of the microbiological, physicochemical, and organoleptic stability of a specimen, since low water activity retards autohydrolysis and microbiological growth. OBJECTIVE: The main purpose of this investigation was to test the stability of powdered raw materials for a maximum holding time of 8 days through water activity measurements. Thus microbiological, physicochemical, and organoleptic measurements were carried out in parallel and simultaneously to experimentally establish a relationship between the status of the water activity of processed raw materials and the microbiological, physicochemical, and organoleptic results. METHODS: The raw materials were stored for a maximum holding time of 8 days, in accordance with United States Pharmacopeia monographs. For all the raw materials tested, water activity measurements were performed using the dew point chilled-mirror method on days 0, 3, and 8. On days 0 and 8, microbiological, physicochemical, and organoleptic assessments were performed. RESULTS: It was established that under these storage conditions, the processed raw materials exhibited water activity below 0.60 during the entire holding time. However, there were statistically significant differences in water activity levels between days 0, 3, and 8 (ANOVA P < 0.05). Despite observing statistically significant differences between days, the microbiological, physicochemical, and organoleptic features were within specification at those water activity levels below 0.60. CONCLUSIONS: Water activity below 0.60 does not allow the growth of microorganisms, and the organoleptic and physicochemical features remain unperturbed. The results indicate that water activity can be used as an indicator of the microbiological load and chemical stability of the raw materials tested. HIGHLIGHTS: This research provides evidence that corroborates that water activity status may be used as a reliable indicator for the microbiological burden and physicochemical features of pharmaceutical material during stability studies.

Glyoxyl-disulfide agarose: a tailor-made support for site-directed rigidification of proteins.

A new strategy has been developed for site-directed immobilization/rigidification of genetically modified enzymes through multipoint covalent attachment on bifunctional disulfide-glyoxyl supports. Here the mechanism is described as a two-step immobilization/rigidification protocol where the enzyme is directly immobilized by thiol-disulfide exchange between the ß-thiol of the single genetically introduced cysteine and the few disulfide groups presented on the support surface (3 µmol/g). Afterward, the enzyme is uniquely rigidified by multipoint covalent attachment (MCA) between the lysine residues in the vicinity of the introduced cysteine and the many glyoxyl groups (220 µmol/g) on the support surface. Both site-directed immobilization and rigidification have been possible only on these novel bifunctional supports. In fact, this technology has made possible to elucidate the protein regions where rigidification by MCA promoted higher protein stabilizations. Hence, rigidification of vicinity of position 333 from lipase 2 from Geobacillus thermocatenulatus (BTL2) promoted a stabilization factor of 33 regarding the unipunctual site-directed immobilized derivative. In the same context, rigidification of penicillin G acylase from E. coli (PGA) through position ß201 resulted in a stabilization factor of 1069. Remarkably, when PGA was site-directed rigidified through that position, it presented a half-life time of 140 h under 60% (v/v) of dioxane and 4 °C, meaning a derivative eight times more stable than the PGA randomly immobilized on glyoxyl-disulfide agarose. Herein we have opened a new scenario to optimize the stabilization of proteins via multipoint covalent immobilization, which may represent a breakthrough in tailor-made tridimensional rigidification of proteins.

Human C3 mutation reveals a mechanism of dense deposit disease pathogenesis and provides insights into complement activation and regulation.

Dense deposit disease (DDD) is a severe renal disease characterized by accumulation of electron-dense material in the mesangium and glomerular basement membrane. Previously, DDD has been associated with deficiency of factor H (fH), a plasma regulator of the alternative pathway (AP) of complement activation, and studies in animal models have linked pathogenesis to the massive complement factor 3 (C3) activation caused by this deficiency. Here, we identified a unique DDD pedigree that associates disease with a mutation in the C3 gene. Mutant C(3923ΔDG), which lacks 2 amino acids, could not be cleaved to C3b by the AP C3-convertase and was therefore the predominant circulating C3 protein in the patients. However, upon activation to C3b by proteases, or to C3(H2O) by spontaneous thioester hydrolysis, C(3923ΔDG) generated an active AP C3-convertase that was regulated normally by decay accelerating factor (DAF) but was resistant to decay by fH. Moreover, activated C(3b923ΔDG) and C3(H2O)(923ΔDG) were resistant to proteolysis by factor I (fI) in the presence of fH, but were efficiently inactivated in the presence of membrane cofactor protein (MCP). These characteristics cause a fluid phase-restricted AP dysregulation in the patients that continuously activated and consumed C3 produced by the normal C3 allele. These findings expose structural requirements in C3 that are critical for recognition of the substrate C3 by the AP C3-convertase and for the regulatory activities of fH, DAF, and MCP, all of which have implications for therapeutic developments.

Coexistence of closed and open conformations of complement factor B in the alternative pathway C3bB(Mg2+) proconvertase.

Complement factor B (fB) circulates in plasma as a proenzyme that, upon binding to C3b in the presence of Mg(2+), is cleaved by factor D to produce Ba and Bb fragments. Activated Bb remains bound to C3b organizing the alternative pathway C3 convertase (C3bBb). Recently, we have visualized the stable C3bB(Ni(2+)) proconvertase using electron microscopy, revealing a large conformational change of the C3b-bound fB likely exposing the fD-cleavage site. In contrast, the crystal structure of the proconvertase formed by human fB and the cobra venom factor reveals fB in the closed conformation of the proenzyme. In this study, we have used single-particle electron microscopy and image processing to examine the C3bB(Mg(2+)) proconvertase. We describe two C3bB(Mg(2+)) conformations, one resembling cobra venom factor, likely representing the loading state of fB to C3b, and another identical with C3bB(Ni(2+)). These data illustrate the coexistence of C3b-bound fB in closed and open conformations that either exist in equilibrium or represent structural transitions during the assembly of the C3bB proconvertase.

The disease-protective complement factor H allotypic variant Ile62 shows increased binding affinity for C3b and enhanced cofactor activity.

Mutations and polymorphisms in the gene encoding factor H (CFH) have been associated with atypical haemolytic uraemic syndrome, dense deposit disease and age-related macular degeneration. The disease-predisposing CFH variants show a differential association with pathology that has been very useful to unravel critical events in the pathogenesis of one or other disease. In contrast, the factor H (fH)-Ile(62) polymorphism confers strong protection to all three diseases. Using ELISA-based methods and surface plasmon resonance analyses, we show here that the protective fH-Ile(62) variant binds more efficiently to C3b than fH-Val(62) and competes better with factor B in proconvertase formation. Functional analyses demonstrate an increased cofactor activity for fH-Ile(62) in the factor I-mediated cleavage of fluid phase and surface-bound C3b; however, the two fH variants show no differences in decay accelerating activity. From these data, we conclude that the protective effect of the fH-Ile(62) variant is due to its better capacity to bind C3b, inhibit proconvertase formation and catalyze inactivation of fluid-phase and surface-bound C3b. This demonstration of the functional consequences of the fH-Ile(62) polymorphism provides relevant insights into the complement regulatory activities of fH that will be useful in disease prediction and future development of effective therapeutics for disorders caused by complement dysregulation.

Functional basis of protection against age-related macular degeneration conferred by a common polymorphism in complement factor B.

Mutations and polymorphisms in complement genes have been linked with numerous rare and prevalent disorders, implicating dysregulation of complement in pathogenesis. The 3 common alleles of factor B (fB) encode Arg (fB(32R)), Gln (fB(32Q)), or Trp (fB(32W)) at position 32 in the Ba domain. The fB(32Q) allele is protective for age-related macular degeneration, the commonest cause of blindness in developed countries. Factor B variants were purified from plasma of homozygous individuals and were tested in hemolysis assays. The protective variant fB(32Q) had decreased activity compared with fB(32R). Biacore comparison revealed markedly different proenzyme formation; fB(32R) bound C3b with 4-fold higher affinity, and formation of activated convertase was enhanced. Binding and functional differences were confirmed with recombinant fB(32R) and fB(32Q); an intermediate affinity was revealed for fB(32W). To confirm contribution of Ba to binding, affinity of Ba for C3b was determined. Ba-fB(32R) had 3-fold higher affinity compared with Ba-fB(32Q). We demonstrate that the disease-protective effect of fB(32Q) is consequent on decreased potential to form convertase and amplify complement activation. Knowledge of the functional consequences of polymorphisms in complement activators and regulators will aid disease prediction and inform targeting of diagnostics and therapeutics.

3D structure of the C3bB complex provides insights into the activation and regulation of the complement alternative pathway convertase.

Generation of the alternative pathway C3-convertase, the central amplification enzyme of the complement cascade, initiates by the binding of factor B (fB) to C3b to form the proconvertase, C3bB. C3bB is subsequently cleaved by factor D (fD) at a single site in fB, producing Ba and Bb fragments. Ba dissociates from the complex, while Bb remains bound to C3b, forming the active alternative pathway convertase, C3bBb. Using single-particle electron microscopy we have determined the 3-dimensional structures of the C3bB and the C3bBb complexes at approximately 27A resolution. The C3bB structure shows that fB undergoes a dramatic conformational change upon binding to C3b. However, the C3b-bound fB structure was easily interpreted after independently fitting the atomic structures of the isolated Bb and Ba fragments. Interestingly, the divalent cation-binding site in the von Willebrand type A domain in Bb faces the C345C domain of C3b, whereas the serine-protease domain of Bb points outwards. The structure also shows that the Ba fragment interacts with C3b separately from Bb at the level of the alpha'NT and CUB domains. Within this conformation, the long and flexible linker between Bb and Ba is likely exposed and accessible for cleavage by fD to form the active convertase, C3bBb. The architecture of the C3bB and C3bBb complexes reveals that C3b could promote cleavage and activation of fB by actively displacing the Ba domain from the von Willebrand type A domain in free fB. These structures provide a structural basis to understand fundamental aspects of the activation and regulation of the alternative pathway C3-convertase.

Genetic deficiency of complement factor H in a patient with age-related macular degeneration and membranoproliferative glomerulonephritis.

Age-related macular degeneration (AMD) and membranoproliferative glomerulonephritis type II (MPGN2) are dense deposit diseases that share a genetic association with complement genes and have complement proteins as important components of the dense deposits. Here, we present the case of a 64-year-old smoker male who developed both AMD and MPGN2 in his late 50s. The patient presented persistent low plasma levels of C3, factor H levels in the lower part of the normal range and C3NeF traces. Genetic analyses of the CFH, CFB, C3, CFHR1-CFHR3 and LOC387715/HTRA1 genes revealed that the patient was heterozygote for a novel missense mutation in exon 9 of CFH (c.1292 G>A) that results in a Cys431Tyr substitution in SCR7 of the factor H protein. In addition, he was homozygote for the His402 CFH allele, heterozygote for the Ser69 LOC387715 allele, homozygote for the Arg32 (BFS) CFB allele, heterozygote for the Gly102 (C3F) C3 allele and carried no deletion of the CFHR1/CFHR3 genes. Proteomic and functional analyses indicate absence in plasma of the factor H allele carrying the Cys431Tyr mutation. As a whole, these data recapitulate a prototypical complement genetic profile, including a partial factor H deficiency and the presence of major risk factors for AMD and MPGN2, which support the hypothesis that these dense deposit diseases have a common pathogenic mechanism involving dysregulation of the alternative pathway of complement activation.

Mixed ion exchange supports as useful ion exchangers for protein purification: purification of penicillin G acylase from Escherichia coli.

A support having similar amounts of carboxymethyl and amino groups has been prepared and evaluated as an ion exchanger. It has been found that this support was able to adsorb a high amount of protein from a crude extract of proteins (approximately 55%) at pH 5. Moreover, it was able to adsorb approximately 60% of the protein that did not become adsorbed on supports bearing just one kind of ionic groups. The use of divalent cations reinforced the adsorption of proteins on these supports. These results suggest that the adsorption of proteins on supports bearing almost neutral charge is not driven by the existence of opposite charges between the adsorbent and the biomacromolecule but just by the possibility of forming a high number of enzyme-support ionic bonds. This support has been used to purify the enzyme penicillin G acylase (PGA) from Escherichia coli. PGA was not significantly adsorbed at any pH value on either amino- or carboxyl-activated supports, while it can be fully adsorbed at pH 5 on this new carboxyl-amino matrix. Thus, we have been able to almost fully purify PGA from crude extracts with a very high yield by using these new supports.

Genetic modification of the penicillin G acylase surface to improve its reversible immobilization on ionic exchangers.

A new mutant of the industrial enzyme penicillin G acylase (PGA) from Escherichia coli has been designed to improve its reversible immobilization on anionic exchangers (DEAE- or polyethyleneimine [PEI]-coated agarose) by assembling eight new glutamic residues distributed homogeneously through the enzyme surface via site-directed mutagenesis. The mutant PGA is produced and processed in vivo as is the native enzyme. Moreover, it has a similar specific activity to and shows the same pH activity profile as native PGA; however, its isoelectric point decreased from 6.4 to 4.3. Although the new enzyme is adsorbed on both supports, the adsorption was even stronger when supports were coated with PEI, allowing us to improve the enzyme stability in organic cosolvents. The use of restrictive conditions during the enzyme adsorption on anionic exchangers (pH 5 and high ionic strength) permitted us to still further increase the strength of adsorption and the enzyme stability in the presence of organic solvents, suggesting that these conditions allow the penetration of the enzyme inside the polymeric beds, thus becoming fully covered with the polymer. After the enzyme inactivation, it can be desorbed to reuse the support. The possibility to improve the immobilization properties on an enzyme by site-directed mutagenesis of its surface opens a promising new scenario for enzyme engineering.

Chemical modification of protein surfaces to improve their reversible enzyme immobilization on ionic exchangers.

The enzyme penicillin G acylase (PGA) is not adsorbed at pH 7 on DEAE- or PEI-coated supports, neither is it adsorbed on carboxymethyl (CM)- or dextran sulfate (DS)-coated supports. The surface of the enzyme was chemically modified under controlled conditions: chemical amination of the protein surface of carboxylic groups (using soluble carbodiimide and ethylendiamine) and chemical succinylation (using succinic anhydride) of amino groups. The full chemical modification produced some negative effects on enzyme stability and activity, although partial modification (mainly succinylation) presented negligible effects on both enzyme features. The chemical amination of the protein surface permitted the immobilization of the enzyme on CM- and DS-coated support, while the chemical succinylation permitted the enzyme immobilization on DEAE- and PEI-coated supports. Immobilization was very strong on these supports, mainly in the polymeric ones, and dependent on the degree of modification, although the enzymes still can be desorbed after inactivation by incubation under drastic conditions. Moreover, the immobilization on ionic polymeric beds allowed a significant increase in enzyme stability against the inactivation and inhibitory effects of organic solvents, very likely by the promotion of a certain partition of the organic solvent out of the enzyme environment. These results suggest that the enrichment of the surface of proteins with ionic groups may be a good strategy to take advantage of the immobilization of industrial enzymes via ionic exchange on ionic polymeric beds.

Improved stabilization of chemically aminated enzymes via multipoint covalent attachment on glyoxyl supports.

The surface carboxylic groups of penicillin G acylase and glutaryl acylase were chemically aminated in a controlled way by reaction with ethylenediamine via the 1-ethyl-3-(dimethylamino-propyl) carbodiimide coupling method. Then, both proteins were immobilized on glyoxyl agarose. In both cases, the immobilization of the chemically modified enzymes improved the enzyme stability compared to the stability of the immobilized but non-modified enzyme (by a four-fold factor in the case of PGA and a 20-fold factor in the case of GA). The chemical modification presented a deleterious effect on soluble enzyme stability. Therefore, the improved stability should be related to a higher multipoint covalent attachment, involving both the lysine amino groups and also the new amino groups chemically introduced on the enzyme. Moreover, the lower pK(a) of the new amino groups permitted to immobilize the enzyme under milder conditions. In fact, the aminated proteins could be immobilized even at pH 9, while the non-modified enzymes could only be immobilized at pH over 10.