HK1220408B - Lipid sterilisation method - Google Patents

Description

Lipid sterilization method

Technical Field

The present invention relates to a method for sterilizing a phospholipid suspension, which can be used to prepare an ultrasound contrast agent precursor comprising phospholipid-stabilized perfluorobutane microbubbles. The method provides sterility assurance without excessive thermal degradation of the phospholipids. The method is also suitable for industrial-scale production. Also provided are methods for preparing a kit and an ultrasound contrast agent incorporating the sterilization method of the present invention.

Background of the Invention

Ultrasound contrast agents based on phospholipid-stabilized microbubbles of perfluorocarbons (eg, perfluoropropane or perfluorobutane) are well known in the art [see, eg, Wheatley et al. J. Drug Del. Sci. Technol., 23(1), 57-72 (2013)].

WO 97/11683 discloses that phospholipids undergo hydrolysis and addresses the need to stabilize such compositions during autoclaving (page 6, first entire paragraph). WO 97/11683 teaches the use of a stabilizing buffer having a pH less than or equal to 9.5 in an attempt to prevent degradation of the phospholipids during autoclaving.

WO 97/29782 teaches that room temperature stable ultrasound contrast agent precursors can be prepared by freeze-drying perfluorocarbon microbubbles in the presence of a freeze-drying stabilizer selected from sucrose, maltose, trehalose, raffinose, or stachyose, preferably sucrose. WO 97/29782 also teaches that the content of phosphatidylserine phospholipids used to stabilize such contrast agents can be reduced during heat sterilization.

WO 99/08715 discloses a method for preparing a pharmaceutical composition comprising an aqueous dispersion of gas-containing vesicles, wherein the membrane comprises an amphiphilic membrane-forming material, the method comprising:

(i) generating a dispersion containing gas vesicles from a mixture comprising an amphiphilic membrane-forming material;

(ii) freeze-drying the dispersion containing the gas-containing vesicles;

(iii) reconstituting the lyophilized product of step (ii) with a sterile aqueous liquid to produce an aqueous dispersion containing gas vesicles; and

(iv) treating the aqueous dispersion product of step (i) or step (iii) or the lyophilized product of step (ii) to produce an aqueous dispersion of gas-containing vesicles that is substantially free of aggregation and sterile.

EP 1228770A1 teaches a method for preparing a lyophilized ultrasound contrast agent containing gas microbubbles, wherein a lyophilized precursor vial is sealed under reduced pressure in the headspace gas. The reduced pressure is believed to help control the particle size of the aqueous microbubble composition formed after reconstitution of the precursor vial.

Feshitan et al. [J.Coll.Interf.Sci., 329, 316-324 (2009)] reported that lipid-coated microbubbles were stable after centrifugation compared to surfactant-coated microbubbles, and the lipid shell was highly viscous and relatively impermeable to air. Feshitan et al. reported that perfluorobutane microbubbles in the size range of 4-5 microns were stable for at least 2 days, but tended to fragment into smaller-sized microbubbles (1-2 microns) after 2 weeks.

There is still a need for a method for preparing ultrasonic lyophilized precursors of reagents incorporated with phospholipids that controls the sterility of the product. Such phospholipid sterilization methods need to be amenable to industrial scale production and produce precursor products with uniform vial content and safety characteristics that meet regulatory requirements (e.g., USP and ICH guidelines).

Summary of the Invention

Phospholipid-stabilized gas microbubble ultrasound contrast agents intended for parenteral administration to mammals need to be prepared in a sterile form. Terminal sterilization is not a suitable technology for such contrast agents, so a sterile form of phospholipid suspension is required to allow aseptic production. Industrial-scale production of contrast agents requires that large volumes (multi-liters) must be sterilized.

The present inventors have discovered that thermal degradation of the phospholipids in hydrogenated egg phosphatidylserine can occur during autoclaving. Therefore, phospholipids are heat-sensitive and can degrade when heated at high temperatures for extended periods. Furthermore, if excessive degradation occurs during sterilization attempts, the desired ultrasonic properties of the gas microbubbles can be compromised. Thus, achieving thorough sterilization without excessive thermal degradation of the phospholipids presents a problem.

This problem is exacerbated at the multi-liter scale, as the larger volume makes it more difficult to ensure uniform heat distribution throughout the system—with the risk that any localized overheating or prolonged exposure will lead to higher levels of phospholipid degradation.

In a standard autoclave sterilization process, typically performed in a jacketed steel vessel, heat is transferred to the bulk material by passing a heat medium (usually steam) through a jacket outside the vessel, which is isolated from the interior of the sterilization system. Thus, such systems utilize sensible heat to transfer the energy required to achieve acceptable sterilization. However, in this case, the endpoints of the sterilization system, such as the inlet, sterile filter for venting, and the vessel cap, receive heat at a later point in time than the bulk material, which is closer to the heat source. All components of the closed system must be sterilized, and this often results in very high heat loads (and _F0 values) on the bulk material before the sterile barrier at the endpoint reaches an acceptable minimum level.

Therefore, with standard heating protocols, the total heat load and thus the chemical degradation of the phospholipids can become unacceptably high.Therefore, there is a need to heat/cool the entire sterilization system (ie the volume contained between the sterile barriers) quickly and uniformly.

The present invention provides a solution to this problem. The method of the present invention involves injecting steam into the phospholipid suspension itself and/or into the headspace above the suspension. Utilizing latent heat (steam heating) for heat transfer is much more efficient than sensible heat from, for example, an external jacket, because a higher amount of energy is released in a shorter period of time. Furthermore, steam injection into the headspace enables rapid heating of endpoints, such as inlets, sterile filters, and vessel lids, thereby reducing the overall heat load on the bulk material. Using headspace steam heating offers the following advantages:

Detailed Description of the Invention

In a first aspect, the present invention provides a method for sterilizing a phospholipid suspension, comprising:

(i) mixing the phospholipids together with propylene glycol in an aqueous biocompatible carrier in a jacketed container to obtain an aqueous phospholipid suspension; and

(ii) autoclaving the aqueous phospholipid suspension from step (i), wherein the autoclaving is capable of achieving an _F0 value greater than 15 for all parts of the sterilization system, and wherein heating comprises, in addition to sensible heat from heating the vessel jacket, adding steam to:

(a) the headspace of the container of step (i); or

(b) the aqueous phospholipid suspension of step (i); or

(c) a combination of (a) and (b);

(iii) cooling the hot suspension from step (ii) to 15-30° C. to obtain a sterile phospholipid suspension;

The phospholipid is hydrogenated egg phosphatidylserine (H-EPS).

The term "sterilization" has its conventional meaning and refers to the process of eliminating microorganisms to obtain a sterile, pyrogen-free composition. The term "autoclaving" has its conventional meaning and refers to a specific sterilization method using superheated steam sterilization. Autoclaving and other sterilization methods are described in "Achieving Sterility in Medical and Pharmaceutical Products" by N. H. Alls (CRC Press, 1994).

The term "sterilization system" refers to the volume contained within the sterilization barriers (ie, inlet and outlet) of the autoclave body.

Assurance of sterility is often described by the term _F0 , defined as "the amount of time, in minutes, equivalent to that imparted to the product by the sterilization process at 121°C" (FDA "Proposed Rule" June 1, 1976(b), Part 212.3). During an autoclave sterilization cycle, the _F0 value is calculated as:

F ₀ = Δt∑10 ^(T-Tb)/Z

in:

△t is the measurement interval,

T is the heating temperature (the temperature measured by the sensor),

Tb is 121.1°C (the specified temperature for steam pasteurization), and

Z is the temperature constant for the logarithmic change in sterilization capacity and is usually set at 10°C.

During the autoclave cycle, even at low temperatures, _F0 will therefore increase with time, but the rate of increase is very temperature dependent.According to USP/EP, the minimum requirement for an acceptable _F0 as an assurance of sterility is _F0 >15.

The term "headspace" has its conventional meaning and refers to the gas phase inside a container and above the liquid and/or solid contents of the container (in this case the aqueous phospholipid suspension).

The term "aqueous phospholipid suspension" refers to a suspension of phospholipids in an aqueous solvent, the aqueous solvent including water and/or a water-miscible solvent. The aqueous solvent is a suitable biocompatible carrier. The term "biocompatible carrier" refers to a fluid, particularly a liquid, that makes the composition physiologically tolerable, i.e., can be administered to a mammal without toxicity or undue discomfort. Suitable biocompatible carriers are injection vehicles, such as sterile, pyrogen-free water for injection; aqueous solutions such as saline (which can advantageously be balanced so that the final product for injection is isotonic); aqueous buffer solutions containing biocompatible buffers (e.g., phosphate buffer); aqueous solutions of one or more tonicity-adjusting substances (e.g., salts of plasma cations with biocompatible counterions), sugars (e.g., glucose or sucrose), sugar alcohols (e.g., sorbitol or mannitol), glycols (e.g., glycerol), or other non-ionic polyol materials (e.g., polyethylene glycol, propylene glycol, etc.). Preferred biocompatible carriers are pyrogen-free water for injection (WFI), isotonic saline, and phosphate buffer. Aqueous suspensions therefore suitably do not include water-immiscible organic solvents.

Throughout this application, the terms "comprising" or "including" have their conventional meaning and mean that the agent or composition must have the listed essential features or components, but other features or components may additionally be present. The term "comprising" includes as a preferred subset "consisting essentially of," which means that the composition has the listed components, but no other features or components are present.

Preferred embodiments

In the method of the first aspect, steam is preferably added to the headspace of the vessel in step (i). Thus, while direct injection of steam into the liquid suspension is possible, the addition of the headspace imparts a more controlled heating effect - since injection into the liquid can result in significant local degradation.

The method of the first aspect is preferably carried out so as to achieve an _F0 value of at least 15 and no greater than 25. Thus, the inventors have determined that longer autoclaving heating times (where _F0 reaches 30) lead to excessive degradation of phospholipids. This, in turn, adversely affects the stability of the PFB microbubbles produced therefrom.

Thermal degradation of phosphatidylserine is believed to occur as shown in Scheme 1:

Solution 1

in:

PS = phosphatidylserine;

PA = phosphatidic acid;

S = serine;

FFA = free fatty acids;

LA = lyso-PA;

LS = hemolysis-PS.

The main reaction is the hydrolysis of PS to PA and S, as shown in Scheme 2:

Option 2

wherein each R＝nC ₁₆ chain.

Without wishing to be bound by theory, it is believed that increased degradation means increased levels of PA, an acid, which is more negatively charged than PS (the parent phospholipid), and therefore the charge balance and electrostatic repulsion of the phospholipid "shell" that stabilizes the microbubbles may be affected.

As is clear from Scheme 1, the amount of PS in the suspension after autoclaving relative to the total amount of PS and related substances (i.e., the sum of PS, PA, S, FFA, lyso-PA, and lyso-PS) is a direct measure of degradation. As is evident from Example 4, the percentage of PS relative to the total amount of PA and related substances should preferably be at least 68%, more preferably at least 73%, and most preferably at least 75%. Lower PS levels have been shown to result in inferior microbubble properties.

In the method of the first aspect, the mass ratio of phospholipid to propylene glycol is preferably 1:1.5 to 1:2.5, more preferably 1:2.

In the method of the first aspect, the volume of the aqueous phospholipid suspension of step (i) is preferably in the range of 20 to 80 liters, more preferably 30 to 70 liters, most preferably 40 to 60 liters.

Sterilization of phospholipid mixtures, particularly at multi-liter scales suitable for industrial production, is a challenge. This is because the aforementioned thermal degradation competes with the heating necessary to ensure sterilization. Sterile filtration of lipid suspensions has been shown to reduce the yield and quality of microvesicles produced in subsequent production steps, and gamma irradiation is not a suitable in-process sterilization technique. Consequently, acceptable sterility assurance is difficult to achieve, as larger volumes require longer heating times to obtain the required sterility assurance (F ₀ greater than 15).

To achieve this, the present invention uses steam supplied directly to the headspace of the vessel. In the method of the first aspect, clean steam (as defined in GMP guidelines CFR Title 21, Section 211) is preferably used in step (ii). The method of the first aspect preferably further comprises applying a vacuum pulse or multiple vacuum pulses to the headspace of the vessel of step (i) to remove the headspace gas before adding steam to the headspace. The headspace gas is primarily air, which is a good insulator, and removing the headspace gas before injecting steam greatly improves subsequent heat transfer.

In the method of the first aspect, the vessel of step (i) is suitably a jacketed vessel (e.g. as shown in FIG1 ) so that the heating of step (ii) further comprises adding hot steam to the vessel jacket - i.e. the heating method of the present invention is supplemented with a more conventional heating method to maximize heat transfer. A vacuum is cyclically drawn from the head space of the vessel through the media inlet ( FIG1 , point A). With the first vacuum pulse, most of the air in the head space is drawn out and then the pressure increases again due to evaporation of the liquid in the vessel. A new vacuum cycle is applied to draw out the remaining mixture of air and water vapor. Most preferably, three vacuum pulses are applied before the steam injection. These pulses remove all air from the head space of the vessel and from system endpoints such as the air filter ( FIG1 , point B) and the media inlet.

The injection of clean steam is preferably carried out several times during the cycle, for example at the beginning and end of the autoclave cycle. The first injection helps to heat the top of the container (Figure 1, point C), which is usually a steel structure with a high heat capacity. If it is not heated with steam, it will cause a significant extension of the autoclave cycle and subsequently excessive degradation of the lipid suspension body, as shown in Example 1.

Typically, three temperature sensors are included in the sterilization system; one in the lipid suspension body (Figure 1, point D), one just outside the sterile barrier (valve) at the media inlet (Figure 1, point E), and one just outside the sterile barrier at the sterile exhaust filter (Figure 1, point F). For an acceptable sterilization cycle, the requirement is that all sensors have an F _value greater than 15.

Subsequent clean steam injection is preferably performed when the liquid phase reaches an _F0 greater than 15 (a set point _F0 greater than 16 is typically applied to allow for uncertainty). This injection rapidly sterilizes the surfaces of the vessel top, air filter, and media inlet, and prevents prolonged exposure to sensible heat from the vessel jacket and further prevents lipid degradation in the bulk material, as shown in Example 2. When an _F0 greater than 16 is reached in the air filter and media inlet, the product is rapidly cooled by applying cooling water to the vessel jacket.

Condensate from the clean steam added during the first part of the autoclave cycle also compensates for some of the water that evaporates from the H-EPS suspension during the autoclave cycle.

H-EPS can be purchased from NOF Corporation, Amagasaki-Shi, Hyogo, Japan. The analysis and quantification of the phospholipid components of egg phosphatidylserine and the ultrasound contrast agent Sonazoid ^™ have been described by Hvattum et al. [J. Pharm. Biomed. Anal., 42, 506-512 (2006)]. Such analysis is also applicable to the method of the present invention.

In a second aspect, the present invention provides a method for preparing an ultrasound contrast agent precursor comprising a lyophilized composition of perfluorobutane microbubbles stabilized with hydrogenated egg phosphatidylserine in sucrose, wherein the method comprises:

(i) implementing the method of the first aspect to obtain a sterile aqueous suspension of hydrogenated egg phosphatidylserine;

(ii) forming microbubbles of perfluorobutane in the suspension from step (i) to obtain an aqueous suspension of perfluorobutane microbubbles stabilized by hydrogenated egg phosphatidylserine;

(iii) diluting the suspension from step (ii) with a sterile aqueous sucrose solution to obtain a final sucrose concentration of 5-20% w/v;

(iv) dispensing an aliquot of the composition from step (iii) into the vial to obtain a filled vial;

(v) freeze-drying the filled vials from step (iv);

(vi) backfilling the headspace gas of the freeze-dried vial from step (v) with perfluorobutane;

(vii) Seal each vial from step (vi) with a cap.

Preferred embodiments of the method of step (i) of the second aspect are also described in the first aspect (above).

The term "precursor" refers to a convenient form or kit form of a contrast agent which is designed such that upon reconstitution it readily forms the desired ultrasound contrast agent.

The term "contrast agent" has its conventional meaning in the field of in vivo medical imaging and refers to an agent in a form suitable for mammalian administration that helps provide clearer images in the area or organ of interest compared to imaging obtained from a mammalian subject alone. The term "subject" refers to a mammalian body, preferably an intact mammalian body, more preferably a living human subject. The phrase "form suitable for mammalian administration" refers to a composition that is sterile, pyrogen-free, does not contain compounds that produce toxic or side effects, and is formulated at a biocompatible pH (pH of about 4.0 to 10.5). This composition does not contain microparticles that may cause embolic risks in vivo and is formulated so that it does not precipitate when in contact with biological fluids (e.g., blood). This composition also contains only biocompatible excipients and is preferably isotonic.

Like other in vivo imaging agents, contrast agents are designed to have minimal pharmacological effects on the mammalian subject being imaged. Preferably, the contrast agent can be administered into the mammalian body in a minimally invasive manner, i.e., without substantial health risks to the mammalian subject when administered by a qualified medical professional. Such minimally invasive administration is preferably intravenous administration into a peripheral vein of the subject, without the need for local or general anesthesia.

The term "microbubble" has its conventional meaning in the field of in vivo ultrasound imaging and refers to a gas microbubble with a diameter generally between 0.5 and 10 microns. When the size is greater than 7 microns, there is a significant risk of retention (embolism) in the pulmonary capillaries. Suitable microbubbles are similar in size to red blood cells, which makes them show similar properties in the microvessels and capillaries of the mammalian body [Sirsi et al., Bubble Sci. Eng. Technol., 1 (1-2), 3-17 (2009)].

Suitable microbubbles of the present invention are stabilized by the phospholipids present in hydrogenated egg phosphatidylserine (H-EPS). The phospholipids present in H-EPS are mainly phosphatidylserine and phosphatidic acid [Hvattum et al., J. Pharm. Biomed-Anal., 42, 506-512 (2006)].

Perfluorobutane ("PFB") has its standard chemical meaning and is also referred _{to as perflubutane in the context of medical use. The chemical formula of perfluoro-n-butane is CF3CF2CF2CF3 or C4F10 , and its boiling} point is -2.2°C. Commercial perfluoro-n- _butane contains a small amount (typically 2-4%) of the perfluoroisobutane isomer, _CF3CF ( _CF3 ) _CF3 .

In the method of the second aspect, the sucrose concentration in step (iii) is preferably 8-12% w/v, more preferably about 10% w/v, and most preferably 92 mg/mL. Sucrose, as a lyoprotectant/cryoprotectant, has been shown to form a matrix in which lipid-stabilized PFB microbubbles are trapped in the freeze-dried sucrose of the precursor, such that the size and concentration of the microbubbles are predetermined and are not affected by the reconstitution procedure adopted by the end user. Thus, upon reconstitution of the precursor vial with a suitable aqueous medium, the sucrose dissolves, releasing the phospholipid-stabilized PFB microbubbles [Sontum, Ultraso. Med. Biol., 34(5), 824-833 (2008)]. The use of sugars as lyoprotectants or cryoprotectants is well known in the art and is described, for example, in Solis et al. [Int. J. Pharmaceut., 396, 30-38 (2010)] and references therein.

The term "filled vial" refers to a filled vial into which an aliquot of the composition has been dispensed, i.e., a dispensing vial. The vial is not physically full, as the vial capacity is typically 10 mL, and the dispensed volume is approximately 2 mL—leaving a headspace of gas above the composition. The term "headspace gas" has its conventional meaning and refers to the gas above the lyophilized composition in the vial.

In the method of the second aspect, the freeze-dried precursor composition is sterile.

In the method of the second aspect, the batch size preferably ranges from 500 to 80,000, more preferably from 1,000 to 50,000, most preferably from 5,000 to 45,000 vials to be filled with the contrast agent precursor. An ideal batch size is about 35,000 to 40,000 vials.

The preparation of microbubbles in step (ii) of the method of the second aspect can be carried out by various methods well known in the art, including:

(i) ultrasonic treatment;

(ii) high shear emulsification;

(iii) membrane emulsification;

(iv) coaxial electrohydrodynamic atomization (CEHDA); and

(v) microfluidic devices;

(vi) Inkjet printing.

These methods are described by Stride et al. [Soft Matter, 4, 2350-2359 (2008)] and references therein. Further details of microbubble preparation methods are provided by Unnikrishnan et al. [Am. J. Roentgenol, 199 (2), 292-299 (2012)].

Stride et al. (supra) reported that inkjet printing is more suitable for liquid-filled particles and is therefore less suitable for the PFB microbubbles of the present invention. Ultrasonication is not preferred because it tends to give a wide range of bubble sizes, and microfluidics suffers from slow production speeds, making it unsuitable for industrial production [Wang et al., Ultraso. Med. Biol., 39(5), 882-892 (2013)].

The preferred production method of the present invention is high shear emulsification, preferably using a rotor-stator mixer-because this gives a high degree of size control, is suitable for large-scale production, and provides a static size distribution. In the presence of a perfluorocarbon (PFB), microbubbles are formed by stirring a sterile, pyrogen-free lipid suspension for a few seconds. Example 3 provides a rotor-stator microbubble preparation method. Rotor-stator mixers are described in Rodgers et al. [Chem. Eng. Res. Rev., 90 (3), 323-327 (2012)] and Emulsion Formation and Stability [T. F. Tadros (ed.), Wiley VCH (2013)]-see in particular Pacek et al., Chapter 5, Emulsification in Rotor-Stator Mixers, pp. 127-167.

The method of the present invention is particularly suitable for industrial-scale production, for example, for production batches of up to 100,000, typically about 35,000 to 40,000, vials of contrast agent precursor. In such large-scale industrial production, consistency from vial to vial, i.e., minimizing vial-to-vial variation, is important. Such batch sizes require multi-liter volumes of the phospholipid suspension described in the first aspect (above).

A preferred ultrasound contrast agent and precursor of the present invention is Sonazoid ^™ (GE Healthcare AS), formerly known as NC100100, as described by Sontum [Ultraso. Med. Biol., 34(5), 824-833 (2008)].

Suitable vials and closures for use in the method of the first aspect are pharmaceutical grade, suitable for freeze drying, and commercially available in large quantities. Flat-bottomed vials are preferred because they increase heat transfer between the vials and the freeze dryer shelf. The closure is preferably suitable for freeze drying and designed to allow vapor to escape from the vial, making it possible to plug the vial before the freeze dryer is opened and unloaded.

Sucrose is also commercially available - pharmaceutical grade should be used. Pharmaceutical GMP grade perfluorobutane is available from F2 Chemicals Limited.

In a third aspect, the present invention provides a method for preparing a kit for preparing an ultrasound contrast agent, comprising:

(i) carrying out the method of the second aspect to obtain a vial comprising a contrast agent precursor as defined therein; and

(ii) preparing a container of a sterile aqueous medium suitable for reconstitution of said precursor vial from step (i) to obtain said contrast agent;

The ultrasound contrast agent comprises a suspension of perfluorobutane microbubbles stabilized by hydrogenated egg phosphatidylserine in the sterile aqueous medium.

In a preferred embodiment of the method of the second aspect, the precursor and contrast agent in the kit preparation method of the third aspect are as described in the first and second aspects (above).

The term "kit" has its conventional meaning in the field of in vivo imaging and refers to the imaging agent itself or its precursor, which is provided with all the necessary components to prepare the contrast agent of interest in a form suitable for mammalian administration (as defined above). Such kits are designed to have a shelf life of several weeks or months so that the clinician can prepare the imaging agent at the appropriate time and opportunity to image the mammalian subject of interest. This is generally more convenient for the clinician because, once prepared, the contrast agent itself has a very short shelf life. Supplying the kit means that the clinician has 24/7 access to the contrast agent, whether needed or not. The kit may appropriately include a "package insert" to define the kit and its components, provide detailed information on how to use the kit, together with patient safety information, and details of the manufacturer. Such kits generally represent the preferred form of commercial products designed to provide ultrasound contrast agents.

The sterile aqueous solution of step (ii) of the third aspect is preferably a "biocompatible carrier" as defined above. More preferably, it is sterile water for injection. Most preferably, the sterile aqueous solution has a total concentration of free (i.e., non-chelated) aluminum, barium, magnesium, calcium, and zinc ions of less than 100 μM, ideally less than 50 μM.

The kit of the second aspect preferably further comprises a vent filter (5 μm) spike, such as a spike (Codan GmbH & Co, Germany). The kit is reconstituted with an aqueous solution by the spike, followed by manual mixing for about 1 minute to obtain a milky, uniform dispersion. The dose of contrast agent for imaging the subject is then drawn into a syringe through the filter spike. The purpose of the "spike" is to remove any foreign particles or clumps - which would otherwise be difficult to detect by visual inspection because the dispersion in the vial is opaque.

In a fourth aspect, the present invention provides a method for preparing an ultrasound contrast agent, comprising:

(i) carrying out the method of the second aspect to obtain a vial containing a contrast agent precursor as defined therein; and

(ii) reconstituting the precursor vial from step (i) with a sterile aqueous solution;

The ultrasound contrast agent comprises a suspension of perfluorobutane microbubbles stabilized by hydrogenated egg phosphatidylserine in the sterile aqueous medium.

In a preferred embodiment of the method of the second aspect, the precursor and contrast agent in the contrast agent preparation method of the fourth aspect are as described in the first and second aspects (above). The method of the fourth aspect is preferably implemented using the kit described in the third aspect.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a schematic diagram of a typical system for autoclaving liquid products such as lipid suspensions; a 50 L steel jacketed vessel. Points A, B, C, D, E, F, and G are the temperature sensors for the media inlet, sterile air filter, vessel top, heating jacket, bulk product, inlet, and air filter, respectively.

Figure 2 shows the temperature obtained by the bulk product sensor over time for an autoclave cycle using steam injection and a heating jacket (top) and an autoclave cycle using only a heating jacket (bottom). The arrows indicate the start of heating/steam injection and the point at which _F0 is greater than 16. The double arrows indicate the time span over which the bulk product temperature reaches greater than 80°C.

Figure 3 shows the relationship between temperature (Figures 3a and 3b) and _F0 values (Figures 3c and 3d) versus time during an autoclave sterilization cycle using an initial steam injection/heating jacket, with (Figures 3b and 3d) and without (Figures 3a and 3c) secondary steam injection, when the bulk product reaches an F0 _greater than 16.

The present invention is illustrated by the following non-limiting examples detailed below. Example 1 shows the effectiveness of the steam injection technique of the present invention for achieving a rapid temperature rise, thereby avoiding prolonged heating of the phospholipid suspension ( FIG. 2 ). In the conventional method, without steam injection, the total heat load, expressed as the time above 80° C., before reaching an F ₀ above 16 is greater than 85 minutes, compared to approximately 35 minutes when steam injection is used at the beginning of the cycle. Table 1 shows the effect of this difference on the PS level after autoclaving. When steam injection is used for autoclaving, the PS content after autoclaving is 83%, which is reduced to 75% when steam injection is not used for autoclaving.

Example 2 demonstrates the effectiveness of steam injection at the end of the autoclave cycle (Figures 3a-d), where the _F0 value of the sterile barrier (inlet and air filter) increases almost immediately to greater than 16, thereby allowing the total heat load (time greater than 80°C) to be further reduced from approximately 35 minutes to approximately 30 minutes (Figures 3a-d). Example 3 provides the preparation of PFB microbubbles stabilized with H-EPS (the contrast agent Sonazoid ^™ ) using rotor-stator mixing.

Example 4 demonstrates the effect of the heating time of the autoclave cycle on the lipid composition of the contrast agent Sonazoid ^™ . As can be seen in Table 2, PS levels decrease upon heating, accompanied by an increase in PA concentration. Table 3 shows that the properties of the ultrasound contrast agent deteriorate significantly between autoclave cycles of 23 minutes (F ₀ = 25) and 30 minutes (F ₀ = 30), causing the latter to fail to meet specifications for several standards. This demonstrates the need for careful control of the heating cycle and the methods according to the present invention.

abbreviation

FD: freeze-dried;

GMP: Good Manufacturing Practice;

H-EPS: hydrogenated egg phosphatidylserine;

ICH: International Conference on Harmonization;

i.v.: intravenous;

Min: minute;

PA: phosphatidic acid;

PFB: perfluorobutane;

PS: phosphatidylserine;

Rpm: rotations per minute;

WFI: Water for injection.

Example 1: Heating using headspace steam injection: Method I.

To investigate the effect of steam injection on the heat load acting on the phospholipids and the subsequent effect on lipid degradation; four autoclave cycles were performed, comparing a standard cycle with jacket heating only to a cycle with steam injection into the vessel headspace at the beginning of the sterilization procedure.

A jacketed stainless steel pressure vessel (large volume autoclave) from Novaferm (50 L) was used. 125 g H-EPS was added to 25 L of WFI containing 10 mg/ml propylene glycol and hydrated at 60° C. with stirring for 20 minutes before the start of the autoclave cycle. A sensor monitored the temperature in the bulk lipid suspension and continuously calculated F _0. For the steam injection cycle, a vacuum was drawn from the headspace of the container before steam injection in three cycles. Clean steam injection was implemented at the start of the autoclave cycle. Cooling with water (ambient temperature) was started when the F ₀ value was greater than 16. Two identical autoclave cycles were repeated using the same H-EPS raw material with and without the steam injection procedure.

The results of the relationship between the temperature and time of the main product are shown in Figure 2.

The phospholipid composition of the autoclaved suspension preparation was analyzed according to Hvattum et al. [J. Pharm. Biomed. Anal., 42, 506-512 (2006)]. The results are summarized in Table 1:

Table 1: PS and PA content in lipid suspensions autoclaved to F ₀ = 16 using conventional jacket heating with and without steam injection into the vessel headspace. Values are given as percentage of the total amount of PS and PA.

sample cycle PS (%) PA (%) 1 No steam injection 76.3 23.7 2 No steam injection 73.2 26.8 3 Steam injection 82.6 17.4 4 Steam injection 82.5 17.5

Figure 2 and Table 1 demonstrate the effectiveness of the steam injection technique of the present invention in achieving a rapid temperature rise, thereby avoiding prolonged heating of the phospholipid suspension. Using conventional methods, without steam injection, the total heat load, expressed as time above 80°C, was greater than 85 minutes before _F0 reached greater than 16, compared to approximately 35 minutes when steam injection was used at the beginning of the cycle. In fact, using conventional jacket heating alone, sterilization plateaus were not established at all.

Table 1 shows the impact of this difference on PS and PA levels after autoclaving. Notably, as the heat load increases, the PS content decreases significantly, while the PA content increases. The PS content (PS + PA percentage) after autoclaving is 83% when steam injection is used for autoclaving, but decreases to 75% when steam injection is not used for autoclaving.

Example 2: Heating using headspace steam injection: Method II.

To investigate the effect of steam injection on the heat load acting on the phospholipids, autoclave cycles were performed comparing cycles using steam injection at the start of the sterilization process with jacket heating and steam injection to cycles where steam was injected into the headspace when the _F0 of the bulk material reached 16.

A jacketed stainless steel pressure vessel (large volume autoclave) from Diesel (77 L) was used. 250 g of H-EPS was added to 50 L of WFI containing 10 mg/ml propylene glycol and hydrated at 60°C with stirring for 20 minutes before the autoclave cycle began. Temperature probes were used to monitor the temperature within the phospholipid suspension and at the sterile barrier at the media inlet and air filter, as shown in Figure 1. The _F0 value was continuously calculated for each sensor.

Heating was initiated by adding conventional house steam (1.25 barG, 124°C) to the jacket. Three 40-second vacuum pulses were applied, and 45-second steam injection was applied through the media inlet at 2.35 barG and 137°C. The vacuum pulse/steam injection procedure was repeated when the bulk suspension had an _F0 value greater than 16. Cooling of the jacket with water (ambient temperature) was initiated when the calculated _F0 value of both sterile barriers was greater than 16.

The results of the temperature and F ₀ values versus time for the three sensors are shown in Figures 3a–d.

These results demonstrate the effectiveness of steam injection at the end of the autoclave cycle, where the _F0 value of the sterile barrier (inlet and air filter) increases almost immediately to greater than 16, thus allowing the total heat load imposed on the phospholipid suspension (expressed as time greater than 80°C) to be further reduced from approximately 35 minutes to approximately 30 minutes.

Example 3: Preparation of Sonazoid ^™ drug product by rotor-stator mixing and lyophilization.

Sterile H-EPS suspension was prepared as detailed in Example 4. For each batch (A, B, and C), Sonazoid ^™ drug product was produced as detailed below. The suspension of Part A (500 mL) was transferred to a round-bottom flask with a conical neck. The flask was equipped with a glass jacket with a temperature-controlled inlet and outlet and connected to a water bath maintained at 25°C.

The rotor-stator mixing shaft is inserted into the solution. In order to avoid gas leakage, the space between the neck wall and the mixing shaft is sealed with a specially designed metal plug, which is connected to the gas inlet/outlet to match, for regulating gas capacity and control pressure. The gas outlet is connected to a vacuum pump, and the solution is degassed for 1 minute. Subsequently, the atmosphere of perfluoro-n-butane gas is applied through the gas inlet. The solution was homogenized 10 minutes under 23000rpm, and the rotor-stator mixing shaft was kept so that the opening is slightly above the liquid surface.

Obtain a white milky dispersion, transfer it to a sealable container and rinse with perfluoro-n-butane. Then 440g of the dispersion is transferred to a flotation/separation container (85mm in diameter) for multi-stage size and concentration adjustment. Allow the dispersion to rest for 400 minutes, then release 250mL through the bottom valve. This step is repeated 4 times. The microbubble concentration is subsequently determined by Coulter counting. Based on the results of the process analysis, the final freeze-dried dispersion is subsequently adjusted to a fixed target microbubble concentration of 1.7% v/v. Concentration adjustment is performed with WFI containing 184mg/mL sucrose, and the WFI in the final dispersion contains 92mg/mL sucrose. For each batch of A, B and C, 2mL of the final dispersion is transferred to a 10mL sterile glass vial with a freeze-drying stopper (N=30). Freeze drying is performed using an Amsco Finn Aqua Lyovac GT6 pilot freeze dryer according to the cycle described below.

stage Temperature (℃) Time (h) Pressure (μbar) freezing -60 2 environment Primary drying -32 72 52 Secondary drying 34 38 20

Example 4: Effect of autoclaving time on the phospholipid content of Sonazoid ^™ .

To determine the degradation level of the Sonazoid ^™ product and the impact on critical mass attributes of degradation level, sterilization of a hydrated phospholipid suspension in a large volume autoclave was studied for 15, 23, and 30 minutes.

A jacketed Buchiglasuster micro autoclave (1 L) was used. 4 g of H-EPS sodium was added to 800 mL of WFI containing 10 mg/mL propylene glycol and mixed at 60°C and 200 rpm for 20 minutes before the autoclave cycle began. A temperature sensor placed in the body of the suspension was used to continuously monitor the temperature of the suspension. The jacket was placed in a hot silicone oil bath, maintained at 160°C during heating, and adjusted to 145°C when the suspension reached the set temperature of 121°C. In batches A, B and C, the suspension was kept at this temperature for 15, 23 and 30 minutes, respectively (the measured temperature varied between 121 and 124°C). At the end of the cycle, cooling water at 20°C was applied to the jacket. After reaching room temperature, the sterile H-EPS suspension was used for the production of Sonazoid ^™ as described in detail in Example 3.

The drug product Sonazoid ^™ was prepared according to Example 3 in three batches A, B and C, which differed in the autoclave time (minutes at 121° C.) used for the sterilization of the phospholipid suspension. The phospholipid components of suspensions A, B and C were analyzed by TLC according to Hvattum et al. [J. Pharm. Biomed. Anal., 42, 506-512 (2006)]. The concentration and size of the microbubbles, the acoustic attenuation efficiency at 3.5 MHz and the pressure stability (as a percentage of the attenuation efficiency at 3.5 MHz after 60 seconds of pressure application at 120 mmHg) were determined as described by Sontum [Ultraso. Med. Biol., 34 (5), 824-833 (2008)]. The results are summarized in Tables 2 and 3 below:

These results demonstrate the effect of autoclaving heating time on the lipid composition of the contrast agent Sonazoid ^™ . As can be seen in Table 1, PS levels decreased upon heating, accompanied by an increase in the concentrations of PA and other related substances. Table 2 shows that the properties of the ultrasound contrast agent deteriorated significantly between autoclaving cycles of 23 minutes (F ₀ = 25) and 30 minutes (F ₀ = 30), causing the latter to fail the specifications of several standards. This demonstrates the need for careful control of the heating cycle according to the present invention.

From these data, it is clear that in order to produce Sonazoid with acceptable properties, the amount of PS, calculated as a percentage of the total amount of PS and related substances, should be at least 68%. The PA content should be less than 15% relative to the total amount of PS and related substances. Lower PS levels will result in reduced yield and poorer microbubble properties.

Table 2: Lipid composition of lipid suspension after autoclaving. Identified lipid components and degradation products are detailed in Scheme 1. _IX is the sum of unidentified related degradation products. Data are expressed in mg/mL.

sample PS PA FFA L-PS L-PA S sum PS/Sum A (15 minutes) 3.65 0.55 0.15 0.23 0.03 0.16 0.05 4.8 0.76 B (23 minutes) 3.24 0.70 0.20 0.32 0.05 0.20 0.06 4.8 0.68 C (30 minutes) 3.10 0.74 0.23 0.37 0.06 0.21 0.06 4.8 0.65

Table 3: Critical Quality Attributes of Drug Products

parameter A (15 minutes) B (23 minutes) C (30 minutes) Volume (% v/v) 0.98% 0.99% 0.56%* Median particle size 2.88μm 2.78μm 2.4μm 3.5MHz attenuation 17.9dB/cm 18.8dB/cm 9.5dB/cm* Attenuation after pressurization 92% 94% 89.3%*

*: Below specification.

Claims (14)

1. A method for sterilizing a phospholipid suspension, comprising: (i) The phospholipids and propylene glycol are mixed in a jacketed container in an aqueous biocompatible carrier to obtain an aqueous phospholipid suspension; and (ii) Autoclave the aqueous phospholipid suspension from step (i), wherein the autoclaving enables the _F0 value of all parts of the sterilization system to be between 15 and 25, and wherein, in addition to the sensible heat from heating the container jacket, the heating also includes adding steam to the top space of the container from step (i). (iii) Cool the hot suspension from step (ii) to 15-30°C to obtain a sterile phospholipid suspension; The phospholipid mentioned therein is hydrogenated lecithinylserine. 2. The method of claim 1, wherein adding steam to the top space is performed multiple times during the autoclaving cycle. 3. The method of any one of claims 1 to 2, further comprising applying a vacuum to the top space of the container in step (i) before adding steam to the top space. 4. The method of any one of claims 1 to 2, wherein the content of phosphatidylserine in the sterile phospholipid suspension of step (iii) is at least 68% relative to the sum of phosphatidylserine, phosphatidic acid, serine, free fatty acids, lysophosphatidic acid and lysophosphatidylserine. 5. The method of any one of claims 1 to 2, wherein the concentration of phosphatidylserine in the sterile phospholipid suspension of step (iii) is less than 15% relative to the sum of phosphatidylserine, phosphatidic acid, serine, free fatty acids, lysophosphatidic acid and lysophosphatidylserine. 6. The method of any one of claims 1 to 2, wherein phosphatidylserine is degraded into phosphatidic acid during sterilization, and the combined concentration of phosphatidylserine and phosphatidic acid in the sterile phospholipid suspension in step (iii) is 5 mg/mL. 7. The method according to any one of claims 1 to 2, wherein the mass ratio of phospholipid to propylene glycol is 1:2. 8. The method of any one of claims 1 to 2, wherein clean steam is used in step (ii). 9. The method of any one of claims 1 to 2, wherein the volume of the aqueous phospholipid suspension in step (i) is in the range of 20 to 80 L. 10. A method for preparing an ultrasound contrast agent precursor, said precursor comprising a lyophilized composition of perfluorobutane microbubbles stabilized in sucrose with hydrogenated lecithin serine; wherein the method comprises: (i) To obtain a sterile aqueous suspension of hydrogenated lecithin acylserine by performing the method of any one of claims 1 to 9; (ii) Forming perfluorobutane microbubbles in the suspension from step (i) to obtain an aqueous suspension of perfluorobutane microbubbles stabilized by hydrogenated lecithin acylserine. (iii) Dilute the suspension from step (ii) with sterile aqueous sucrose solution to obtain a final sucrose concentration of 5-20% w/v; (iv) Dispense an equal portion of the composition from step (iii) into a vial to obtain a full vial; (v) Freeze-dry the filled vials from step (iv); (vi) Backfill the headspace gas from the freeze-dried vial from step (v) with perfluorobutane; (vii) Seal each vial from step (vi) with a cap. 11. A method for preparing a kit for preparing an ultrasound contrast agent, comprising: (i) performing the method of claim 10 to obtain a vial containing the contrast agent precursor as defined in claim 10; and (ii) Prepare a container suitable for reconstructing the precursor vial from step (i) to obtain a sterile aqueous medium of the contrast agent; wherein the ultrasound contrast agent comprises a suspension of perfluorobutane microbubbles stabilized with hydrogenated lecithin serine in the sterile aqueous medium. 12. The method of claim 11, wherein the sterile aqueous solution is water for injection containing a total concentration of less than 100 μM of free aluminum, barium, magnesium, calcium and zinc ions. 13. A method for preparing an ultrasound contrast agent, comprising: (i) performing the method of claim 10 to obtain a vial containing the contrast agent precursor as defined in claim 10; and (ii) Reconstitute the precursor vial from step (i) with a sterile aqueous solution; The ultrasound contrast agent comprises a suspension of perfluorobutane microbubbles stabilized with hydrogenated lecithin serine in the sterile aqueous medium. 14. The method of claim 13, wherein the method is performed using the kit defined in claim 11.