Strategies to Reduce Reconstitution Time of Lyophilized Biotherapeutics.

Lyophilized biotherapeutics with high protein concentration may have long reconstitution times, which pose an inconvenience to the end user. This report describes 2 approaches that lead to reduction of reconstitution time: (1) incorporation of tert-butyl alcohol (TBA) in the prelyophilization formulation and (2) decreased headspace pressure in the final lyophilized vial. Cakes made from prelyophilization formulations containing a range of TBA concentrations were physically characterized. The stability of antibodies with TBA in the liquid and lyophilized states was evaluated under stress conditions. Reconstitution time was minimized (>50% reduction) at a TBA concentration of 5% w/v. Reduced headspace pressure in the lyophilized vial demonstrated greater than 50% reduction in reconstitution time at headspace pressures of less than 50 Torr.

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.

High shear rheology and anisotropy in concentrated solutions of monoclonal antibodies.

The high shear rheology of three concentrated solutions of immunoglobulin G1 monoclonal antibodies (mAb1, mAb2, and mAb3), differing only in their complementarity determining regions, was characterized using rotary and capillary rheometry. The more viscous solutions (mAb1 and mAb3) showed non-Newtonian behavior at high shear rates exhibiting both shear thinning and appreciable normal stress differences (NSDs) in the shear rate range γ = 10 to 10(4) s(-1) . The rheograms were retraced after γ is increased and decreased, suggesting reversible self-associations under shear. In contrast, mAb2 solutions showed Newtonian behavior up to γ = 6 × 10(4) s(-1) . The critical shear stress τc , corresponding to the onset of the reduction in the viscosity η, is a measure of mAb equilibrium cluster strength and increased rapidly with concentration for the high viscosity mAb solutions above 100 mg/mL. In addition, decreasing the temperature from 20°C to 5°C increased η at low γ, but shear-thinning was enhanced and its onset occurred at a lower γc . Using an Arrhenius model η = A exp(Ea /kT), the activation energy for viscous flow Ea was found to decrease for mAb1 solutions as γ was increased from 10 to 10(4) s(-1) , suggesting mAb cluster disruption or rearrangement under shear. In contrast, for mAb2, this Ea remained constant in the γ range. Finally, mAb1 and mAb3 solutions showed appreciable NSDs, with their N1 > 0 scaling linearly with γ in the range 10(3) to 10(4) s(-1) , whereas their |N2 /N1 | was less than 0.25 in this region. These suggest anisotropy and deformation of their solution microstructure toward the extensional quadrant of the flow at high γ. In contrast, the NSDs for mAb2 were close to zero indicating that the solution microstructure under shear is practically isotropic.

Covalently tethered transforming growth factor beta in PEG hydrogels promotes chondrogenic differentiation of encapsulated human mesenchymal stem cells.

Methods to precisely control growth factor presentation in a local and sustained fashion are of increasing interest for a number of complex tissue engineering applications. The cytokine transforming growth factor beta (TGFß) plays a key role in promoting the chondrogenic differentiation of human mesenchymal stem cells (hMSCs). Traditional chondrogenic approaches utilize soluble delivery, an approach with limited application for clinical translation. In this work, we introduce a reactive thiol onto TGFß and covalently tether the growth factor into poly(ethylene glycol) (PEG) hydrogels using a photoinitiated thiol-acrylate polymerization mechanism. We demonstrate the bioactivity of thiolated TGFß, before and after polymerization, using a SMAD2 reporter cell line. hMSCs were encapsulated in PEG hydrogels with and without tethered TGFß, and subsequently assayed for glycosaminoglycan and collagen II production as indicators of chondrogenesis. Over a 21-day time course, tethered TGFß promoted chondrogenesis at levels similar to a positive control using solubly dosed growth factor. These results provide evidence that tethered TGFß materials can be successfully used to promote chondrogenic differentiation of MSCs.