Using different proteolytic enzymes to digest antibody and its impact on stability of antibody mimetics

There are opportunities to formulate antibodies as solid-state depots for local therapy, which would minimise large systemic doses that are typically required. We have developed antibody mimetics known as Fab-PEG-Fab (FpF) that display similar binding affinity and functional activity as IgG antibodies. For head-to-head comparison between FpF and IgG, FpF is prepared from the Fabs obtained by enzymatic digestion of IgGs. Here, we report for the first time that using different enzymes to proteolytically digest IgG plays an important role in stability profile of the obtained Fabs leading in different stability profiles of the final conjugated product such as FpF. We prepared an anti-vascular endothelial growth factor (VEGF) FpF from either clinical Fabrani (ranibizumab) or Fabs obtained by enzymatic digestion of bevacizumab (IgG) using immobilised papain and gingisKHANTM (KGP) enzyme. The stability of FpFs was then studied after being lyophilised in comparison with both ranibizumab and bevacizumab. Lyophilisation is being evaluated to produce solid material that can be used for depot fabrication. We observed that using immobilised papain to digest IgG resulted in the heterogenous isomers Fab leading to the preparation of heterogenous FpFbeva-papain mimetic that underwent aggregation during lyophilisation. However, using KGP enzyme generated a homogenous intact Fabbeva-KGP as determined by mass spectral analysis. Interestingly, the FpF mimetics prepared from the homogenous Fabs (Fabrani and Fabbeva-KGP), displayed greater stability compared to their starting bevacizumab and ranibizumab after being lyophilised as determined by DLS analysis. There is a potential to lyophilize FpFs to be used to fabricate solid-state depots

Soluble Papain to Digest Monoclonal Antibodies; Time and Cost-Effective Method to Obtain Fab Fragment

Antigen binding fragments (Fabs) used in research (e.g., antibody mimetics, antibody-drug conjugate, bispecific antibodies) are frequently obtained by enzymatic digestion of monoclonal antibodies using immobilised papain. Despite obtaining pure Fab, using immobilised papain to digest IgG has limitations, most notably slow digestion time (more than 8 h), high cost and limited scalability. Here we report a time and cost-effective method to produce pure, active and stable Fab using soluble papain. Large laboratory scale digestion of an antibody (100 mg) was achieved using soluble papain with a digestion time of 30 min and isolated yields of 55–60%. The obtained Fabs displayed similar binding activity as Fabs prepared via immobilised papain digestion. Site-specific conjugation between Fabs and polyethylene glycol (PEG) was carried out to obtain antibody mimetics FpF (Fab-PEG-Fab) indicating that the native disulphide bond had been preserved. Surface-plasmon resonance (SPR) of prepared FpFs showed that binding activity towards the intended antigen was maintained. We anticipate that this work will provide a fast and less costly method for researchers to produce antibody fragments at large scale from whole IgG suitable for use in research

Non-adiabatic corrections to elastic scattering of halo nuclei

We derive the formalism for the leading order corrections to the adiabatic approximation to the scattering of composite projectiles. Assuming a two-body projectile of core plus loosely-bound valence particle and a model (the core recoil model) in which the interaction of the valence particle and the target can be neglected, we derive the non-adiabatic correction terms both exactly, using a partial wave analysis, and using the eikonal approximation. Along with the expected energy dependence of the corrections, there is also a strong dependence on the valence-to-core mass ratio and on the strength of the imaginary potential for the core-target interaction, which relates to absorption of the core in its scattering by the target. The strength and diffuseness of the core-target potential also determine the size of the corrections. The first order non-adiabatic corrections were found to be smaller than qualitative estimates would expect. The large absorption associated with the core-target interaction in such halo nuclei as Be11 kills off most of the non-adiabatic corrections. We give an improved estimate for the range of validity of the adiabatic approximation when the valence-target interaction is neglected, which includes the effect of core absorption. Some consideration was given to the validity of the eikonal approximation in our calculations.Comment: 14 pages with 10 figures, REVTeX4, AMS-LaTeX v2.13, submitted to Phys. Rev.

Effects of date palm fruit extracts on skin mucosal immunity, immune related genes expression and growth performance of common carp (Cyprinus carpio) fry

The aim of this study was to investigate the effects of date palm fruit extracts (DPFE) on skin mucosal immunity, immune related genes expression and growth performance of fry common carp (Cyprinus carpio). One hundred and twenty specimens (4.06 ± 0.13 g) were supplied and allocated into six aquaria; specimens in three aquaria were fed non-supplemented diet (control) while the fish in the other 3 aquaria were fed with DPFE at 200 ml kg-1. At the end of feeding trial (8 weeks) skin mucus immune parameters (total immunoglobulins, lysozyme, protease and alkaline phosphatase activity) and immune related gene expression (tumor necrosis factor α [tnfa], lysozyme [ly] and interleukin-1-beta, [il1b]) in the head-kidney were studied. The results revealed that feeding carp fry with 200 ml kg-1 DPFE remarkably elevated the three skin mucus immune parameters tested (P 0.05) compared to control fish (fed control diet). Furthermore, growth performance parameters were significantly improved in fry fed DPFE (P < 0.05). More studies are needed to understand different aspects of DPFE administration in fry mucosal immunity. © 2015 Elsevier Ltd

Structure and arrangement of photoreceptors in the retina of big eye kilka, Clupeonella grimmi (Kessler 1877)

The big eye kilka, Clupeonella grimmi, is a marine fish living in depth of 20 to 200 meter of the Caspian Sea. Its eye and retina were processed for histological and SEM studies. Paraffin embedded retina was cut radially and tangentially in 5 µm thickness and stained with hematoxylin and eosin method. The unstained sections were manipulated for SEM image observations. Tangential retinal sections showed irregular arrangement of cones. Five morphologically different types of photoreceptors were distinguished as rods, short single cones, long single cones, twin cones and double cones. The cones were counted in each quadrant of the retina and cone density was determined. The cones showed increment in diameter but reduction in number at anti-clockwise direction without any specific arrangement. Presence of four types of cone cells and their pattern of distribution revealed assistance in near surface color vision and more light capture in dim light of deeper waters as an adaptation to planktivorous feeding habit and deep water living habitat

Fc-fusion mimetics

The Fc-fusion mimetic RpR 2[combining low line] was prepared by disulfide bridging conjugation using PEG in the place of the Fc. RpR 2[combining low line] displayed higher affinity for VEGF than aflibercept. This is caused primarily by a slower dissociation rate, which can prolong a drug at its site of action. RpRs have considerable potential for development as stable, organ specific therapeutics

Quantum back-action in measurements of zero-point mechanical oscillations

Measurement-induced back action, a direct consequence of the Heisenberg Uncertainty Principle, is the defining feature of quantum measurements. We use quantum measurement theory to analyze the recent experiment of Safavi-Naeini et al. [Phys. Rev. Lett. {\bf 108}, 033602 (2012)], and show that results of this experiment not only characterize the zero-point fluctuation of a near-ground-state nanomechanical oscillator, but also demonstrate the existence of quantum back-action noise --- through correlations that exist between sensing noise and back-action noise. These correlations arise from the quantum coherence between the mechanical oscillator and the measuring device, which build up during the measurement process, and are key to improving sensitivities beyond the Standard Quantum Limit.Comment: 11 pages and 4 figure

Feed evaluation

This manual has been produced to standardise some analytical procedures at ILCA and among those NARS scientists who may be interested in the aspects of feed evaluation which the manual attempts to address. The first part of the manual is on determination of voluntary intake, digestion and retention coefficients. It lists feed-intake measurement and collection of faeces and urine and presents procedures on preparing samples for chemical analysis. The second part examines special methods for measuring digestibility. This includes the indicator method, the nylon-bag technique, the nylon-bag procedure, handling nylon-bag data, and the Menke in vitro gas-production technique. The third part summarises the kinetics of digestion and of passage. Flow rates, rumen-evacuation technique, the use of markers to estimate passage rates, and continuous dosing with chromium-mordanted straw are discussed in this part. The fourth part is on the estimation of microbial protein supply using total urine excretion of purine derivatives. This includes sample preparation and mathematical procedure