This work describes the development of polymersome-encapsulated hemoglobin (PEH) self-assembled from biodegradable and biocompatible amphiphilic diblock copolymers composed of poly(ethylene oxide) (PEO), poly(caprolactone) (PCL), and poly(lactide) (PLA). In the amphiphilic diblock, PEO functions as the hydrophilic block, while either PCL or PLA can function as the hydrophobic block. PEO, PCL, and PLA are biocompatible polymers, while the last two polymers are biodegradable. PEH dispersions were prepared by extrusion through 100 nm pore radii polycarbonate membranes. In this work, the encapsulation efficiency of human and bovine hemoglobin (hHb and bHb) in polymersomes was adjusted by varying the initial concentration of Hb. This approach yielded Hb loading capacities that were comparable to values in the literature that supported the successful resuscitation of hamsters experiencing hemorrhagic shock. Moreover, the Hb loading capacities of PEHs in this study can also be tailored simply by controlling the diblock copolymer concentration. In this study, typical Hb/diblock copolymer weight ratios ranged 1.2-1.5, with initial Hb concentrations less than 100 mg/mL. The size distribution, Hb encapsulation efficiency, oxygen affinity (P 50), cooperativity coefficient (n), and methemoglobin (metHb) level of these novel PEH dispersions were consistent with values required for efficient oxygen delivery in the systemic circulation. Taken together, our results demonstrate the development of novel PEH dispersions that are both biocompatible and biodegradable. These novel dispersions show very good promise as therapeutic oxygen carriers.

During the last few decades, liposome encapsulated hemoglobin (LEH) dispersions have been investigated for use as red blood cell (RBC) substitutes. However, the process for formulating LEHs is cumbersome and the composition of the lipid mixture is often complex. This work investigates a simple approach for formulating LEHs from a simple lipid mixture composed of the high phase transition lipid distearoylphosphatidylcholine (DSPC) and cholesterol. In order to improve the circulation half-life and colloidal state of LEHs, the surface of unmodified LEHs was conjugated with poly(ethylene glycol) (PEG-LEHs). The results of this work show that PEG-LEH dispersions exhibited average diameters ranging from 166–195 nm that were colloidally stable for 4–5 months, hemoglobin (Hb) concentrations ranging from 9.6–14 g/dL, methemoglobin levels less than 1%, oxygen affinities (i.e. P50s) ranging from 20–23 mm Hg and cooperativity coefficients ranging from 1.4–2.2. The reactions of PEG-LEHs with physiologically important ligands, such as oxygen (O2), carbon monoxide (CO) and nitric oxide (NO), were also measured. It was observed that PEG-LEHs and RBCs exhibited retarded gaseous ligand binding/release kinetics compared to acellular Hbs. This result provides important insight into the pivotal role that the intracellular diffusion barrier plays in the transport of gases into and out of these structures. Collectively, our results demonstrate that the PEG-LEH dispersions prepared in this study show good potential as a RBC substitute.

Decreasing the timeframe for cell culture process development has been a key goal toward accelerating biopharmaceutical development. Advanced Microscale Bioreactors (ambr™) is an automated micro-bioreactor system with miniature single-use bioreactors with a 10-15 mL working volume controlled by an automated workstation. This system was compared to conventional bioreactor systems in terms of its performance for the production of a monoclonal antibody in a recombinant Chinese Hamster Ovary cell line. The miniaturized bioreactor system was found to produce cell culture profiles that matched across scales to 3 L, 15 L, and 200 L stirred tank bioreactors. The processes used in this article involve complex feed formulations, perturbations, and strict process control within the design space, which are in-line with processes used for commercial scale manufacturing of biopharmaceuticals. Changes to important process parameters in ambr™ resulted in predictable cell growth, viability and titer changes, which were in good agreement to data from the conventional larger scale bioreactors. ambr™ was found to successfully reproduce variations in temperature, dissolved oxygen (DO), and pH conditions similar to the larger bioreactor systems. Additionally, the miniature bioreactors were found to react well to perturbations in pH and DO through adjustments to the Proportional and Integral control loop. The data presented here demonstrates the utility of the ambr™ system as a high throughput system for cell culture process development.

Background The hemoglobin of the Earthworm Lumbricus terrestris (also known as erythrocruorin, or LtEc) is a naturally occurring high molecular weight protein assembly (3.6 MDa) that is extremely stable, resistant to oxidation, and transports oxygen similarly to human whole blood. Therefore, LtEc may serve as an alternative to donated human red blood cells, however, a suitable purification process must be developed to produce highly pure LtEc on a large scale that can be evaluated in an animal model to determine the safety and efficacy of LtEc. Materials and Methods We used tangential flow filtration (TFF), an easily scalable and affordable technique, to produce highly pure LtEc from Earthworms. The purity, yield, methemoglobin level, viscosity, colloid osmotic pressure, O2 binding equilibria, and ligand binding kinetics of the purified LtEc was measured in vitro. The purified LtEc product was then evaluated in hamsters using a hypervolemic infusion model to establish its basic biocompatibility and detect any changes in microcirculatory and systemic parameters. Results TFF was able to produce LtEc with high purity and yield (5–10 grams per 1000 worms). The purified LtEc product did not elicit hypertension or vasoconstriction when infused into hamsters. Conclusion LtEc may be easily purified and safely transfused into hamsters in small amounts (0.5–1.5 g/dL final concentration in blood) without any noticeable side-effects. Therefore, LtEc may serve as a very promising oxygen carrier for use in transfusion medicine.