Low Levels Of Modification Research Articles

Utilizing cross-product prior knowledge to rapidly de-risk chemical liabilities in therapeutic antibody candidates

There is considerable pressure in the pharmaceutical industry to advance better molecules faster. One pervasive concern for protein-based therapeutics is the presence of potential chemical liabilities. We have developed a simple methodology for rapidly de-risking specific chemical concerns in antibody-based molecules using prior knowledge of each individual liability at a specific position in the molecule’s sequence. Our methodology hinges on the development of sequence-aligned chemical liability databases of molecules from different stages of commercialization and on sequence-aligned experimental data from prior molecules that have been developed at Amgen. This approach goes beyond the standard practice of simply flagging all instances of each motif that fall in a CDR. Instead, we de-risk motifs that are common at a specific site in commercial mAb-based molecules (and therefore did not previously pose an insurmountable barrier to commercialization) and motifs at specific sites for which we have prior experimental data indicating acceptably low levels of modification. We have used this approach successfully to identify candidates in a discovery phase program with exclusively very low risk potential chemical liabilities. Identifying these candidates in the discovery phase allowed us to bypass protein engineering and accelerate the program’s timeline by 6 months.

Agricultural modification to vegetation drives presence and abundance of a threatened fossorial legless lizard

AbstractAgricultural modification is one of the main drivers of global biodiversity decline. Vegetation clearance, tillage, grazing, and the application of fertilisers and pesticides have significantly affected native plants and animals. Reptiles can be vulnerable to agricultural practices, and localised declines in reptile biodiversity have been detected. However, the causes of these declines are likely to be many and will vary among reptiles with different life histories. Fossorial reptiles (those that mainly live below ground) are likely to be particularly vulnerable to soil and ground cover disturbance and yet are little studied in the context of agricultural practices. Here, we test the proposition that habitat disturbance caused by agricultural modification is a good predictor of decline or absence of a fossorial lizard. Specifically, we used boosted regression trees to model the relationship between five habitat factors and site abundance of the threatened Pink‐tailed Worm‐lizard (Aprasia parapulchella; Pygopodidae). We found that vegetation characteristics were the best predictors of A. parapulchella abundance. In particular, the relative contributions to our model of percentage cover of native large tussock‐forming grasses and site floristic score were 46% and 33%, respectively. Other variables that contributed to the explanation of abundance were percentage cover of rocks (10%), number of turnable rocks (5%) and relative density of rocks occupied by ants of the size class known to occur with A. parapulchella (5%). Our findings suggest that agricultural management or restoration strategies that promote ground‐layer vegetation characteristic of low levels of modification are likely to be most beneficial for the conservation of this species. Our findings and methods may be applicable to understand and study the presence and abundance of other fossorial or semi‐fossorial reptiles with specialised habitat preferences. Further research is required to establish the generality of our findings with respect to other such species around the world.

Wetting of pristine and functionalized nanocrystalline cellulose

Molecular dynamic simulations were used to investigate the wetting behavior of nanocrystalline cellulose allomorphs. Three 40ns independent MD simulations using CHARMM force field gave droplet profiles, whose contact angles were obtained with LBADSA plugin for ImageJ. Surface hydrophilicity was related to hydroxyl group availability for hydrogen bonding. Oxidation of C6 hydroxyl groups significantly boosts surface hydrophilicity, and even low levels of modification (~7%) leads to contact angles close to zero degrees. These data can be useful for biotechnological applications.

Stable isotope labeling method for the investigation of protein haptenation by electrophilic skin sensitizers.

The risk of contact sensitization is a major consideration in the development of new formulations for personal care products. However, developing a mechanistic approach for non-animal risk assessment requires further understanding of haptenation of skin proteins by sensitizing chemicals, which is the molecular initiating event causative of skin sensitization. The non-stoichiometric nature of protein haptenation results in relatively low levels of modification, often of low abundant proteins, presenting a major challenge for their assignment in complex biological matrices such as skin. Instrumental advances over the last few years have led to a considerable increase in sensitivity of mass spectrometry (MS) techniques. We have combined these advancements with a novel dual-labeling/LC-MS(E) approach to provide an in-depth direct comparison of human serum albumin (HSA), 2,4-dinitro-1-chlorobenzene(DNCB), 5-chloro-2-methyl-4-isothiazolin-3-one (MCI), trans-cinnamaldehyde, and 6-methyl coumarin. These data have revealed novel insights into the differences in protein haptenation between sensitizers with different reaction mechanisms and sensitizing potency; the extreme sensitizers DNCB and MCI were shown to modify a greater number of nucleophilic sites than the moderate sensitizer cinnamaldehyde; and the weak/non-sensitizer 6-methyl coumarin was restricted to only a single nucleophilic residue within HSA. The evaluation of this dual labeling/LC-MS(E) approach using HSA as a model protein has also demonstrated that this strategy could be applied to studying global haptenation in complex mixtures of skin-related proteins by different chemicals.

The comparison of genetic variation in the envelope protein between various immunodeficiency viruses and equine infectious anemia virus

The envelope protein (Env) of lentiviruses such as HIV, SIV, FIV and EIAV is larger than that of other retroviruses. The Chinese EIAV attenuated vaccine is based on Env and has helped to successfully control this virus, demonstrating that envelope is crucial for vaccine. We compared Env variation of the four kinds of lentiviruses. Phylogenetic analysis showed that the evolutionary relationship of Env between HIV and SIV was the closest and they appeared to descend from a common ancestor, and the relationship of HIV and EIAV was the furthest. EIAV had the shortest Env length and the least number of potential N-linked glycosylation sites (PNGS) as well as glycosylation density compared to various immunodeficiency viruses. However, HIV had the longest Env length and the most PNGS. Moreover, the alignment of HIV and SIV showed that PNGS were primarily distributed within extracellular membrane protein gp120 rather than transmembrane gp41. It implies that the size difference among these viruses is associated with a lentivirus specific function and also the diversity of env. There are low levels of modification of glycosylation sites of Env and selection of optimal protective epitopes might be useful for development of an effective vaccine against HIV/AIDS.

Structure and thermal behavior of poly(L‐lactic acid) clay nanocomposites: Effect of preparation method as a function of the nanofiller modification level

AbstractThe structural and thermal characteristics of poly(L‐lactic acid)/layered‐silicate hybrid materials that were produced via two different routes, namely by solvent casting and by melt mixing, were compared in association with the degree of clay modification. Investigation of the produced materials' structure revealed that, at low modification levels, melt blending is necessary in dispersing the amine‐treated clay into the polymer matrix. At intermediate degrees of modification, both techniques are capable of swelling the silicate clay with the solution casting to be a more effective method. Thermal measurements showed that the clay modification level influences significantly the thermal stability of both solution and melt processed hybrids. Moreover, the material derived from melt mixing displayed a higher onset decomposition temperature. The glass transition temperature of the polymer was not significantly affected by the preparation method followed. However, the crystallization process was found to be strongly dependent on both the preparation method and the degree of clay modification. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

Selective Neoglycosylation Increases the Structural Stability of Vicilin, the 7S Storage Globulin from Pea Seeds

The effects of glycosylation on the stability and subunit interactions of vicilin, the major storage protein in pea seeds, were investigated. Glycosylated vicilin derivatives were prepared by alkylation of lysine ϵ-amino groups with various carbohydrates. Average modification levels of 13.4 ± 3.0, 11.1 ± 3.6, 7.5 ± 4.2, and 4.7 ± 0.3 moles of carbohydrate/mol of vicilin were obtained with glucose, galactose, galacturonic acid, and lactose, respectively. Nondenaturing polyacrylamide gel electrophoresis and size-exclusion chromatography indicated that the quaternary structure and hydrodynamic radius of vicilin were not affected by glycosylation at the levels used. We have previously shown that application of hydrostatic pressure causes dissociation of vicilin subunits [C. Pedrosa and S. T. Ferreira (1994) Biochemistry 33, 4046–4055]. Analysis of pressure dissociation data allowed determination of the Gibbs free energy change (ΔGdiss) and molar volume change (ΔVdiss) of dissociation of vicilin subunits. For unmodified vicilin, ΔGdiss = 18.2 kcal/mol and ΔVdiss = −102 ml/mol. Glycosylated vicilin derivatives were significantly stabilized against subunit dissociation, with ΔGdiss of 19.4, 19.2, 20.6, and 22.1 kcal/mol for glucose, galactose, lactose, and galacturonic acid derivatives, respectively. No changes in ΔVdiss were found for the glucose and galactose derivatives, whereas ΔVdiss of −128 and −135 ml/mol, respectively, were found for the lactose and galacturonic acid derivatives. The glycosylated derivatives also appeared more resistant to unfolding by guanidine hydrochloride than unmodified vicilin. Intrinsic fluorescence lifetime measurements showed that glycosylation caused a significant increase in heterogeneity of the fluorescence decay, possibly reflecting increased conformational heterogeneity of glycosylated derivatives relative to unmodified vicilin. These results indicate that the stability and subunit interactions of vicilin may be modulated by mild, selective glycosylation at low modification levels, an effect that may be of interest in the study of other oligomeric proteins.

Efficient in vitro repair of 7-hydro-8-oxodeoxyguanosine by human cell extracts: involvement of multiple pathways.

To investigate the repair of oxidative damage in DNA, we have established an in vitro assay utilizing human lymphoblastoid whole cell extracts and plasmid DNA damaged by exposure to methylene blue and visible light. This treatment has been shown to produce predominantly 7-hydro-8-oxodeoxyguanosine (8-oxodG) in double-stranded DNA at low levels of modification. DNA containing 1. 6 lesions per plasmid is substrate for efficient repair synthesis by cell extracts. The incorporation of dGMP is 2.7 +/- 0.5 times greater than the incorporation of dCMP, indicating an average repair patch of 3-4 nucleotides. Damage-specific nicking occurs within 15 min, while resynthesis is slower. The incorporation of dGMP increases linearly, while the incorporation of dCMP exhibits a distinct lag. Extracts from xeroderma pigmentosum (XP) complementation groups A and B exhibit 25 and 40%, respectively, of the incorporation of dCMP compared with normal extracts, but extracts from an XP-D cell line exhibit twice the activity. These data suggest that the efficient repair of 8-oxodG lesions observed in human cell extracts involves more than one pathway of base excision repair.

Superhelical torsion controls DNA interstrand cross-linking by antitumor cis- diamminedichloroplatinum(II).

Negatively supercoiled, relaxed and linearized forms of pSP73 DNA were modified in cell-free medium by cis-diamminedichloroplatinum(II) (cisplatin). The frequency of interstrand cross-links (ICLs) formed in these DNAs has been determined by: (i) immunochemical analysis; (ii) an assay employing NaCN as a probe of DNA ICLs of cisplatin; (iii) gel electrophoresis under denaturing conditions. At low levels of the modification of DNA (<1 Pt atom fixed per 500 bp) the number of ICLs formed by cisplatin was radically enhanced in supercoiled in comparison with linearized or relaxed DNA. At these low levels of modification, the frequency of ICLs in supercoiled DNA was enhanced with increasing level of negative supercoiling or with decreasing level of modification. In addition, the replication mapping of DNA ICLs of cisplatin was consistent with these lesions being preferentially formed in negatively supercoiled DNA between guanine residues in both the 5'-d(GC)-3' and the 5'-d(CG)-3' sites. Among the DNA adducts of cisplatin the ICL has the markedly greatest capability to unwind the double helix. We suggest that the formation of ICLs of cisplatin is thermodynamically more favored in negatively supercoiled DNA owing mainly to the relaxation of supercoils.

Modification of monoclonal and polyclonal IgG with palladium (II) coproporphyrin I: stimulatory and inhibitory functional effects induced by two different methods

Antibodies conjugated with porphyrins and metalloporphyrins have a great potential for applications in fluorescence or phosphorescence immunoassays as well as in photodynamic therapy, radioimaging and internal radiation therapy of cancer. Here we describe how the new preactivated metalloporphyrin, palladium (II) coproporphyrin I-tetra- N-hydroxysuccinimide ester, can be covalently attached to mouse monoclonal and rabbit anti-human ferritin antibodies. The advantages of the proposed reagent over the previously reported carboxylic porphyrins coupled through carbodiimide activation are indicated. Conformational changes in antibodies caused by each of the two methods were assessed from their binding to the antigen (a probe for the antibody Fv domain) and anti-IgG antibodies probing the global conformation of the C H2 domain in the Fc fragment. Porphyrin coupling through carbodiimide activation resulted in a decrease in both functional activities of modified antibodies even at low levels of modification. In contrast, when the N-hydroxysuccinimide (NHS) derivative of porphyrin was used, enhancement of the antigen-binding affinity of porphyrin-antibody conjugates occurred due to an increase in the conformational mobility (flexibility) of the modified antibodies. The stimulatory effect of conjugation was maximal when one porphyrin molecule was coupled to an antibody molecule. Coupling of NHS-activated porphyrin at pH 7.4, 7.8 and pH 8.5 suggested that the high efficiency of the reaction at pH 8.5 could be attributed predominantly to the formation of antibody aggregates, only 50% of which were covalently cross-linked. The lowest percentage of aggregates in porphyrin-antibody conjugates was found at pH 7.4 and a molar reagent-to-protein ratio in the 10:1–40:1 range. Thus, the use of the NHS-activated carboxylic porphyrin provides a mild, simple and convenient procedure for preparing antibody conjugates with enhanced antigen-binding affinity.

Cooperative self‐assembling in statistical copolymers: a new approach to high‐temperature thermoplastic elastomers

AbstractBased on previous work, where it was shown that 4‐urazoylbenzoic acid (U4A) groups, which are attached statistically to polybutadiene, form ordered supramolecular arrays in the unpolar polymer matrix, the present work describes the synthesis of a new molecular building block capable of self‐assembling in the unpolar polymer matrix. 5‐Urazoylisophthalic acid (U35A) groups attached to 1,4‐polybutadiene chains cause the formation of a thermoplastic elastomer of improved properties. The clusters of functional groups show an endothermic transition. The melting temperature increases for low levels of modification from 130°C up to 190°C. The mechanical properties (stress–strain and dynamic mechanical) reveal excellent rubbery characteristics and a rubbery plateau which is considerably flatter than those of technical grade thermoplastic elastomers. The IR data indicate that the U35A groups are tetrafunctional with respect to supramolecular self‐assembling. Based on detailed knowledge of the structure of the self‐assembled domains in U4A groups, a model is developed which describes the observed material properties qualitatively.

Total proteins and 11S protein fraction (Helianthinin) of sunflower seed (Helianthus annuus L.) — Effect of acetylation and succinylation

AbstractThe total proteins and helianthinin (11S) from sunflower seeds were chemically modified by acetylation and succinylation. The extent of acetylation of the total proteins and helianthinin were 12%, 51%, 52%, 56% and 12%, 36%, 69%, 71%, respectively, while the extent of succinylation were 8%, 21%, 33%, 49% and 10%, 30%, 44%, 61%, respectively. The extent of modification was monitored by the availability of free lysyl residues in the proteins. The ultraviolet absorption maximum shifted to higher wavelengths in total proteins and in helianthinin; there was also an increase in absorbance in the 260 nm wavelength, as a function of increased chemical modification. The sedimentation velocity profile indicated the dissociation of the proteins to low molecular weight fraction (2S) through a 7S component. The dissociation occurred at low modification levels in both total proteins and in helianthinin. There was a gradual red shift and quenching in the fluorescence emission maximum at higher modification levels indicating the denaturation of the proteins as a result of this chemical modification. The change in absorbance as a function of temperature indicates minor changes suggesting that the conformation of the proteins is already altered to significant extents due to the chemical modification.

Succinylation of histone amino groups facilitates transcription of nucleosomal cores

Treatment of nucleosomal cores with succinic anhydride, which modifies preferentially the amino-terminal domains of core histones, takes place without dissociation of the particles. Low levels of modification, which cause small structural effects, are accompanied by substantial increases in the efficiency of the nucleosomal cores as in vitro transcription templates for RNA polymerase II. The transcriptional properties of the succinylated nucleosomal cores are similar to those of the acetylated particles (Piñeiro et al., (1991) Biochem. Biophys. Res. Commun. 177, 370-376), indicating that no specific blocking by acetyl residues is required to facilitate in vitro transcription. Moreover, to obtain a certain level of stimulation, a smaller number of groups has to be modified by succinic than by acetic anhydride.

Pressure-Sensitive Adhesion of Isopropyl-Azodicarboxylate-Modified Polybutadiene to Glass

Abstract The pressure-sensitive adhesion (i.e., adhesive tack) of isopropyl azodicarboxylate modified polybutadiene contacted with glass has been studied using a 180°-peel geometry. Fracture energies were studied as a function of IAD content, contact time, and peeling rate. Additionally, the creep compliance J was measured and related to the peeling response. At low levels of modification, J decreases with increasing IAD content; however, J increases at intermediate IAD levels—indicative of softening in the material. This is thought to be due, at least in part, to a reduction in the entanglement density and is a condition that favors wetting. For example, the elastomer with 33% IAD achieves 46% of its equilibrium bond strength to glass after 2 min of contact while, after the same tc, IAD-29 (with its lower compliance) attains only 26% of its ultimate bond strength. In some circumstances, there is an interesting decrease in the fracture energy as contact time is increased. This is accompanied by a change in the locus of failure from Ig to Im. When failure is Im, the adhesive layer remains flat during peeling and hence, no energy is expended to bend this layer; this results in a lowering of the fracture energy.

Unwinding of supercoiled DNA by cis- and trans-diamminedichloroplatinum(II): influence of the torsional strain on DNA unwinding

The effective unwinding angle, phi, for cis-diamminedichloroplatinum(II) (cis-DDP) and trans-DDP was determined by utilizing high resolution gel electrophoresis and supercoiled phi X174 RF DNA as a substrate. The effective unwinding angle was calculated by equating the reduction in mobility of the DDP-modified DNA to the removal of a number of superhelical turns. The value of the effective unwinding angle for both DDP isomers was greatest at the low levels of DDP bound and decreased with increasing amounts of unwinding agent. The cis-isomer is a better unwinding agent than is the trans-isomer, being nearly twice as effective in unwinding the supercoiled DNA at the DDP levels investigated. A comparison of the magnitude of phi below rb values of 0.005 and those at high levels of binding reveals that the extent of torsional strain in the supercoiled DNA influences the magnitude of the unwinding of the DNA by these complexes. When this method is used in the analysis of the unwinding angle for a covalently bound species on supercoiled DNA, it may provide a more reliable estimate of the magnitude of phi at high degrees of supercoiling and at low levels of modification.

Carbodiimide modification analysis of aminoacylated yeast phenylalanine tRNA: evidence for change in the apex region.

The G- and U-specific reagent, carbodiimide was used to probe the solution structure of aminoacylated yeast phenylalanine tRNA. Both quantitative and qualitative changes in modification were observed when the modification patterns of tRNA-CCA(3'OH), tRNA-CCA(3'NH2) and phe-tRNA-CCA(3'NH2) were compared. Five nucleotides were modified in all cases, D16 and G20 in the D-loop, U33 and Gm34 in the anticodon loop and U47, in the region of the extra arm. Small changes occurred in the D-loop with incorporation of the adenosine analogue manifest as new, low levels of modification of G22 (D-stem) and a loss of sensitivity to Mg+2 in modification of D16. Aminoacylation resulted in new modification of G19, modification of a residue in the T psi CG sequence, and a 2.5-fold increase in modification of G22. Taken together the results show that aminoacylation causes increased exposure of bases in the apex region of the L-shaped molecule where the D- and psi-loops are joined. The effects observed could occur as a consequence of stable or dynamic changes in conformation.

Evidence by chemical modification for the involvement of one or more tryptophanyl residues of bovine antithrombin in the binding of high-affinity heparin.

Tryptophanyl residues of bovine antithrombin were modified with N-bromosuccinimide at near-neutral pH. The reaction was found to be specific for tryptophan at low levels of modification, i.e. when only up to 1--1.3 mol tryptophan/mol protein were oxidized. Further modification led to extensive side reactions. Modification of an average of about one tryptophanyl residue per protein molecule did not affect antithrombin activity measured in the absence of heparin, but decreased the activity assayed in the presence of heparin to about half the value given by unmodified antithrombin. Addition of an excess of high-affinity heparin to a similarly modified antithrombin sample resulted in much smaller circular dichroism, ultraviolet absorption and fluorescence changes than those observed with the intact protein. Modification experiments in the presence of excess high-affinity heparin gave a definitely lower extent of modification than when heparin was excluded. These studies thus reinforce the conclusion from previous spectroscopic analyses that one or more tryptophanyl residues of antithrombin are involved in the binding of high-affinity heparin, presumably by being located at or close to the heparin binding site.