Preliminary studies on the conversion of Tubifex tubifex as food by elvers of Anguilla nebulosa (Gray and Hardwicke)

Elvers of Anguilla nebulosa were reared at 23°C in confined waters and their food intake, growth and conversion efficiency (K1:%) were studied. Elvers weighing 278.33±14.35 mg were fed on an ad libitum diet of the oligochaete worm Tubifex tubifex for 60 days. On an average, the test individuals consumed 99.55±19.81 mg food/elver/day amounting to 35.76% of the initial biomass; during the corresponding period the elvers exhibited a growth rate of 5.03±2.42 mg gain in weight/elver/day which is equivalent to 5.06% of the consumed food. Thus the ‘housekeeping’ of these elvers may be regarded as established. In 50 days, the food required amounts to 0.358±50 = 17.90 times the initially stocked biomass. That is, 1 kg of initially stocked elvers may need 17.90 kg of T. tubifex to produce 0.912 kg of elver meat. A poor conversion ratio of 20 : 1 may be one reason for the slow growth of the elvers

Protein engineering to increase the potential of a therapeutic antibody Fab for long-acting delivery to the eye

To date, ocular antibody therapies for the treatment of retinal diseases rely on injection of the drug into the vitreous chamber of the eye. Given the burden for patients undergoing this procedure, less frequent dosing through the use of long-acting delivery (LAD) technologies is highly desirable. These technologies usually require a highly concentrated formulation and the antibody must be stable against extended exposure to physiological conditions. Here we have increased the potential of a therapeutic antibody antigen-binding fragment (Fab) for LAD by using protein engineering to enhance the chemical and physical stability of the molecule. Structure-guided amino acid substitutions in a negatively charged complementarity determining region (CDR-L1) of an anti-factor D (AFD) Fab resulted in increased chemical stability and solubility. A variant of AFD (AFD.v8), which combines light chain substitutions (VL-D28S:D30E:D31S) with a substitution (VH-D61E) to stabilize a heavy chain isomerization site, retained complement factor D binding and inhibition potency and has properties suitable for LAD. This variant was amenable to high protein concentration (>250 mg/mL), low ionic strength formulation suitable for intravitreal injection. AFD.v8 had acceptable pharmacokinetic (PK) properties upon intravitreal injection in rabbits, and improved stability under both formulation and physiological conditions. Simulations of expected human PK behavior indicated greater exposure with a 25-mg dose enabled by the increased solubility of AFD.v8

Protein engineering to increase the potential of a therapeutic antibody Fab for long-acting delivery to the eye

To date, ocular antibody therapies for the treatment of retinal diseases rely on injection of the drug into the vitreous chamber of the eye. Given the burden for patients undergoing this procedure, less frequent dosing through the use of long-acting delivery (LAD) technologies is highly desirable. These technologies usually require a highly concentrated formulation and the antibody must be stable against extended exposure to physiological conditions. Here we have increased the potential of a therapeutic antibody antigen-binding fragment (Fab) for LAD by using protein engineering to enhance the chemical and physical stability of the molecule. Structure-guided amino acid substitutions in a negatively charged complementarity determining region (CDR-L1) of an anti-factor D (AFD) Fab resulted in increased chemical stability and solubility. A variant of AFD (AFD.v8), which combines light chain substitutions (VL-D28S:D30E:D31S) with a substitution (VH-D61E) to stabilize a heavy chain isomerization site, retained complement factor D binding and inhibition potency and has properties suitable for LAD. This variant was amenable to high protein concentration (>250 mg/mL), low ionic strength formulation suitable for intravitreal injection. AFD.v8 had acceptable pharmacokinetic (PK) properties upon intravitreal injection in rabbits, and improved stability under both formulation and physiological conditions. Simulations of expected human PK behavior indicated greater exposure with a 25-mg dose enabled by the increased solubility of AFD.v8

Bio-Orthogonal Cross-Linking Chemistry Enables <i>In Situ</i> Protein Encapsulation and Provides Sustained Release from Hyaluronic Acid Based Hydrogels

Chemically cross-linked hydrogels are promising systems for protein delivery applications, but their utility may be limited due to the possibility of protein reaction with hydrogel precursors. Herein, a catalyst-free inverse-demand Diels–Alder reaction between tetrazine and norbornene groups was used to demonstrate the bio-orthogonal nature of cross-linking chemistry that is chemically inert to proteins. Tetrazine-modified hyaluronic acid and norbornene-modified polyethylene glycol were used as hydrogel precursors for <i>in situ</i> encapsulation of a model protein, Fab1. Measurement of gelation kinetics demonstrates that network formation and gel stiffness are temperature-dependent but independent of Fab1 concentration. <i>In vitro</i> release testing shows that Fab1 is completely released from the hydrogel matrix over a period of several weeks. Analytical characterization suggests that Fab1 is released without any physical or chemical modifications and retains its antigen binding capacity. Thus, the bio-orthogonal and catalyst-free aqueous phase chemistry enables efficient <i>in situ</i> protein encapsulation in a single step and provides sustained protein release

Polymersomes and Wormlike Micelles Made Fluorescent by Direct Modifications of Block Copolymer Amphiphiles

Wormlike micelles and vesicles prepared from diblock copolymers are attracting great interest for a number of technological applications. Although transmission electron microscopy has remained as the method of choice for assessing the morphologies, fluorescence microscopy has a number of advantages. We show here that when commercially available fluorophores are covalently attached to diblock copolymers, a number of their physicochemical characteristics can be investigated. This method becomes particularly useful for visualizing phase separation within polymer assemblies and assessing the dynamics of wormlike micelles in real time. Near-IR fluorophores can be covalently conjugated to polymers and this opens the possibility for deep-tissue fluorescence imaging of polymer assemblies in drug delivery applications