Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

The overall goal of this procedure is to manufacture a large volume of self assembling, lipid-based oxygen, microbubbles or OMS for intravenous oxygen delivery. This is accomplished using a high shear homogenizer and a gas tight tank to manufacture and serially concentrate oms. The second step is to use modified syringes to extrude and LOM emulsion containing 70 volume percent oxygen.

Next, the emulsion is concentrated by centrifugation to yield a 90 volume percent oxygen foam. The final step is to perform quality control by calculating volume percent oxygen and assessing the particle size distribution using light scattering technology. Ultimately, high shear homogenization produces two liters of OMS containing 90%oxygen by volume.

The main advantage of this technique over existing manufacturing methods like sonication and amalgamation, is that homogenization produces self assembling microbubbles on the order of liters, a volume necessary for large animal testing of therapeutic oxygen delivery. One of the uses of this technique is to treat patients with high hypoxemia because oxygen gas filled microbubbles transfer oxygen to deoxyhemoglobin within seconds and carry three times the oxygen content of blood Visual. Demonstration of this method is important as the depth of the homogenizer head.

The maintenance of a purely oxidated environment and the discernment of concentrated LMS versus the aqueous phase can be difficult to appreciate. The system for manufacturing. Lipid-based oxygen, microbubbles or OMS consists of a holding and concentrating tank or HCT fitted with a single stage mixer, an inline high shear homogenizer, a roller pump to move fluid between the HCT and the homogenizer and a heat exchanger.

To begin the system setup, place a sterilized wide mouthed four liter glass collection vessel fitted with two base ports and three side ports beneath the single stage mixer fit one of the base ports of the HCT port number one with a sterile three eighth inch inner diameter clear tubing, approximately 10 inches long, fitted with a three-way stop cock at the tip for collection of the concentrated emulsion. Next, fit the second base port, port number two with sterile three eighth inch inner diameter tubing, approximately 36 inches in length. Feed this tubing through the roller pump.

The inlet of the high shear homogenizer is fitted with a tee piece that includes two ports. Connect tubing from port number two through the roller pump and connect to the side port of the tee piece. Attach the other port to an oxygen tank using a low flow oxygen gas flow meter.

Connect the outlet port of the high shear homogenizer to the inlet port of an inline heat exchanger maintained at four degrees Celsius. Connect the outlet port of the heat exchanger to the return port of the HCT creating a closed loop system. Attach the oxygen tank via a flow meter to the HCT via port number four.

Lastly, attach a gas composition monitor that is open to atmosphere to the top port of the HCT. To begin this procedure, place 20 grams of GMP one two DYS steer oil, SN glycerol three PHOSPHOCHOLINE or DSPC and 10 grams of cholesterol in the base of the HCT. Add one liter of plasma light A to the HCT and hand stir for one minute, integrating as much lipid as possible into aqueous phase.

Lower the single stage mixer into the aqueous phase, ensuring that the entire mixer head is covered by the aqueous phase To prevent ambient air from contaminating the head space, use rubber seals or tape to ensure that the top of the HCT is gas tight and there are no open side ports. Turn on the gas source attached to port number four and wait until the oxygen fraction of the HCT Headspace reaches greater than 95%Using the single stage mixer mix, the precursor emulsion for five minutes. At 5, 000 RPM, the resulting mixture should appear pale white and contain no visible lipid clumps.

Once mixed, unused lipid water mixture can be stored at four degrees Celsius for up to 30 days before single. Use prime the entire closed loop system with the precursor emulsion by turning on the roller pump at 1.3 liters per minute or LPM. Once the system is primed, keep the pump on at 1.3 LPM to begin manufacture of the LO's.

Turn on the inline high shear homogenizer to 7, 500 RPM. Immediately thereafter, turn on oxygen flow to the inlet portion of the homogenizer at 0.5 LPM. Keep the single stage mixer in the HCT on at 3, 500 RPM.

Oms are formed at the interface of rotor blades and the emulsion screens within the inline homogenizer. Within the first few minutes, the fluid should become visibly more viscous. Run the system for 15 minutes and then turn off the high shear homogenizer and the oxygen inlet to it.

This mitigates phase separation and keeps the product relatively uniform within the HCT. Note that the volume of the gas filled emulsion should increase approximately two to threefold during the serial concentration phase. The 140 milliliter lure lock syringes for collecting the manufactured OMS have previously been modified by withdrawing 100 milliliters of air into the syringe and then sawing off the excess plunger and syringe material above the 140 milliliter mark.

Attach a sterile modified syringe to the stop cock attached to base port number one on the collection vessel used toothed forceps to drop the plunger and draw up 100 milliliters of fluid, tightly capped the syringe repeat until all fluid has been removed. Centrifuge syringes with the capped end oriented downward in a refrigerated bucket centrifuge at 225 times G for 10 minutes. Three layers of material will be apparent after centrifugation, expel and discard the bottom layer of excess cloudy aqueous phase.

The second layer is bright white and contains concentrated oms. Transfer the concentrated foam to a gas impermeable glass syringe using a three-way stop cock to prevent ambient gas contamination. Discard the final layer, which contains free oxygen gas from ruptured oms.

Foam quality can be assessed by calculating gas concentration as follows. A high quality foam will reach a gas concentration of greater than or equal to 90 volume percent as a second quality control size microbubbles by optical light scatter to determine if particle size is within the expected range. It should be noted that a change in homogenization time or formulation might alter bubble size, tightly capped the glass syringes containing concentrated oms.

With a lure lock fitting, syringes can be stored at 22 degrees Celsius, four degrees Celsius or negative 20 degrees Celsius. Colder temperatures may provide enhanced shelf life stability. Using this protocol up to two liters of concentrated OMS can be manufactured in 90 minutes.

Visualization of a 10 microliter sample of OMS by light microscopy revealed spherical OMS as well as a relative posity of lipid debris. When the same sample of OMS was assessed by optical scatter, the mean particle diameter was 2.624 plus or minus 0.332 micrometers. Greater than 90%of OMS were less than 10 micrometers in diameter and the population was poly disperse.

Emulsion viscosity was heavily dependent on gas fraction and therefore on microbubble concentration. Two milliliter aliquots of LOM emulsion at 60, 70 and 90. Volume percent gas were studied using a steady state flow sweep using a 40 millimeter parallel plate.

Geometry as stress was varied from 0.1 to 1000 past gass OMS containing 60 volume percent gas exhibited a radiological profile similar to human blood. The oxygen content of OMS was tested by adding varying volumes of oxygen contained within 60 volume percent OMS to aliquots of desaturated human blood with a known oxygen deficit. The relationship between volume of oxygen added within OMS and the volumetric increase in oxygen content of the blood was 1.053 plus or minus 0.03025.

Suggesting that the OMS tested contain nearly 100%oxygen exhibited few trapped gas pockets, and effectively transfer their entire oxygen payload to human blood in vitro Once mastered, this technique can be used to manufacture two liters of LMS in 90 minutes if it is performed properly and efficiently. While attempting this procedure, it is important to remember to maintain visual inspection of accumulating OMS and to perform quality control upon concentration of LOM foam. Following this manufacturing procedure, OMS can be used for large scale intravenous injection and delivery of therapeutic gases.