KR101168458B1 - Methods for producing storage stable viruses and immunogenic compositions thereof - Google Patents

Abstract

The present invention relates to a method of making a storage stable viral composition. More specifically, the present invention relates to one or more formulations and process steps for providing storage stable viral compositions with storage stability as lyophilized solid compositions or lyophilized liquid compositions.

Description

METHODS FOR PRODUCING STORAGE STABLE VIRUSES AND IMMUNOGENIC COMPOSITIONS THEREOF

The present invention generally relates to the field of virology, viral preparations and process development. More specifically, the present invention relates to a process for preparing storage stable viral compositions with storage stability as lyophilized solid compositions or lyophilized liquid compositions.

Human respiratory syncytial virus (RSV) and parainfluenza virus (PIV), members of the paramyxovirus family, are major pathogens involved in severe respiratory disease in infants and children (Glezen et al., 1981; Chanock et al. , 1992; Martin et al., 1978). Two groups of RSV, Group A (RSV-A) and Group B (RSV-B), are distributed simultaneously during the winter epidemic year after year, but usually appear to be predominantly Group A infections (McConnochie et al., 1990; Stark et al. , 1991). PIV type 3 (PIV-3) is a common cause of bronchiolitis, pneumonia and croup. RSV and PIV-3 together account for up to 30% of all infant and child airway disease inpatients (Crowe, 1995). PIV types 1 and 2 (PIV-1 and PIV-2) are also common causes of croup. RSV has also been reported to cause significant morbidity in people and older adults with compromised immune systems. About 65 million people are infected with RSV every year, causing 160,000 deaths worldwide (Robbins and Freeman, 1988). In the United States alone, 100,000 children are admitted to severe pneumonia and bronchiolitis each year due to RSV infection (Glezen et al., 1986; Katz, 1985). Children who were hospitalized and outpatient for RSV infections in the United States spent over $ 340 million in 1992 alone (Wertz and Sullender, 1992). According to the World Health Organization (Crowe, 1995) and the Allergy and Infectious Disease Research Institute (NIAID) Vaccine Advisory Committee, RSV was second to HIV in vaccine development, and the manufacture of potent PIV (e.g., PIV type 3) vaccines It was ranked as the top 10 vaccines that are considered first.

Therefore, there is an urgent need for safe and effective RSV and / or PIV vaccines that can prevent severe respiratory diseases in infants, children, the elderly and immune-impaired people. The use of live attenuated RSV and / or PIV to control respiratory diseases is one of the more promising solutions. Over the past 20 years, several live attenuated RSV strains have been developed and tested in RSV-serum negative children. The most promising methods of RSV live attenuation are cold-passaged RSV, temperature-sensitive (ts) RSV mutations and cp temperature sensitive (cpts) RSV mutants (Kneyber and Kimpen, 2002). RSV mutants such as cpts-248, cpts-248 / 404, cpts-530 and PIV-3 mutant cp-45 are currently under evaluation and clinical trials in the laboratory.

In addition to the identification and development of potent live attenuated RSV, PIV or RSV / PIV complex immunogenic compositions, methods for preparing storage stable RSV and / or PIV compositions and immunogenic compositions thereof are currently desired. For example, RSV is a heat labile virus that is inactivated within three months during storage at −65 ° C. to −86 ° C. (Hambling, 1964; Wulff et al., 1964; Gupta et al., 1996). Therefore, there is an urgent need for storage stability RSV, PIV or RSV / PIV immunogenic compositions.

In addition, increasing the storage stability of other viral immunogenic compositions has long been recognized as an important goal in improving the effect of vaccines on global health (Melnick and Wallis, 1963; Rasmussen et al., 1973; Ayra, 2001; Hilleman, 1989; Lemon and Milstein, 1994). Therefore, in the virus preparation and processing industry, simple herpes virus, cytomegalovirus, Epstein-Barr virus, varicella zoster virus, mumps virus, measles virus, influenza virus, polio virus ), Rhinovirus, adenovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, Norwalk virus, toga virus, alpha virus, rubella virus, rabies virus, Marburg virus (Marburg virus) ), A method for producing a storage stable virus composition such as Ebola virus, papilloma virus, polyoma virus, metapneumovirus, corona virus, bullous stomatitis virus, Venezuelan equine encephalitis virus, etc. Recognize the need.

Summary of the Invention

The present invention generally relates to storage stable viral compositions and methods of making immunogenic compositions thereof. In certain embodiments, the present invention relates to a method of preparing a storage stable viral composition comprising a respiratory syncytial virus (RSV), parainfluenza virus (PIV), or a combination thereof. More specifically, the present invention relates to one or more formulations and process steps for providing storage stable viral compositions with storage stability as lyophilized solid compositions or lyophilized liquid compositions. In certain embodiments, the present invention relates to one or more formulations and process steps that provide storage stable storage stable RSV, PIV or RSV / PIV compositions as lyophilized solid compositions or lyophilized liquid compositions.

Thus, in certain embodiments, the present invention relates to a method of making a small dose storage stable viral composition. In certain embodiments, the present invention includes the steps of (a) freezing the virus composition below its glass transition temperature within a time of about 60 minutes or less, and (b) lyophilizing the virus composition, wherein the lyophilized virus composition is about A process for the preparation of small dose storage stable viral compositions comprising RSV, PIV or a combination thereof, characterized in being stable for at least one year at a storage temperature of 1 ° C to about 10 ° C. In one example, the glass transition temperature is about −45 ° C. and is reached within a time of about 60 minutes or less. In another example, the glass transition temperature is about −35 ° C. and is reached within about 40 minutes or less. In another example, the glass transition temperature is about −35 ° C., reaching within a time of about 20 minutes or less. In one example, the dose of viral composition is from about 0.2 ml to about 1.0 ml. In certain instances, the viral composition is included in a suitable container means, wherein the container means is further defined as a vial, tube or intranasal spray device. In one example, RSV is further defined as group A RSV (RSV-A), group B RSV (RSV-B) or chimeric recombinant RSV (RSV-AB) comprising one or more antigens of each group A and B, PIV is further limited to PIV type 1 (PIV-1), PIV type 2 (PIV-2), or PIV type 3 (PIV-3).

In certain embodiments, the small dose storage stable viral composition is formulated in a 5.0 mM to about 20 mM phosphate buffer solution containing sodium and / or potassium monobasic and dibasic salts and having a pH of about 6.5 to about 7.8. In another example, the 5.0 mM to about 20 mM phosphate buffer solution further comprises about 0.25 mM to about 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). In another particular embodiment, the 5.0 mM to about 20 mM phosphate buffer solution further comprises about 0.01 mM to about 1 mM magnesium chloride and about 0.01 mM to about 1 mM calcium chloride.

In certain embodiments, the small dose storage stable viral composition is formulated in a 10 mM phosphate buffer solution containing sodium and / or potassium mono and dibasic salts and having a pH of about 6.5 to about 7.8. In another example, the 10 mM phosphate buffer solution further comprises about 0.25 mM to about 25 mM of HEPES. In another specific example, the 10 mM phosphate buffer solution further comprises about 0.01 mM to about 1 mM magnesium chloride and about 0.01 mM to about 1 mM calcium chloride.

In one example, from 5.0 mM to about 20 mM phosphate buffer solution (pH from about 6.5 to about 25 mM) comprising from about 0.25 mM to about 25 mM of HEPES, from about 0.01 mM to about 1 mM of magnesium chloride, and from about 0.01 mM to about 1 mM of calcium chloride. 7.8) includes sucrose, L (+)-glutamic acid, L (+)-glutamic acid monosodium salt, a mixture of L (+)-glutamic acid and L (+)-glutamic acid monosodium salt, human albumin (HA) and / or Further comprises soy peptone. In another embodiment, 5.0 mM to about 20 mM phosphate having a pH of about 6.5 to about 7.8 and comprising about 0.25 mM to about 25 mM HEPES, about 0.01 mM to about 1 mM magnesium chloride, and about 0.01 mM to about 1 mM calcium chloride The buffer solution is about 50 g / L sucrose, about 0.049 mM to about 4.9 mM L (+)-glutamic acid or about 0.049 mM to about 4.9 mM L (+)-glutamic acid monosodium salt or mixtures thereof and about And from 1.0 g / l to about 10.0 g / l HA. In another example, about 1.0 g / l to about 10.0 g / l of HA is replaced with about 50 g / l of soy peptone. As yet another example, a 5.0 mM to about 20 mM phosphate buffer solution at a pH of about 6.5 to about 7.8 may contain about 0.25 mM to about 25 mM HEPES, about 0.01 mM to about 1 mM magnesium chloride, about 0.01 mM to about 1 mM calcium chloride, about 50 g / L sucrose, about 0.049 mM to about 4.9 mM L (+)-glutamic acid or about 0.049 mM to about 4.9 mM L (+)-glutamic acid monosodium salt or mixtures thereof, From about 1.0 g / l to about 10.0 g / l HA and about 50 g / l Soy peptone.

In one example, a 10 mM phosphate buffer solution comprising a pH of about 6.5 to about 7.8 and comprising about 0.25 mM to about 12.5 mM HEPES, about 0.01 mM to about 0.5 mM magnesium chloride and about 0.01 mM to about 0.5 mM calcium chloride About 50 g / l sucrose, about 0.049 mM to about 4.9 mM L (+)-glutamic acid or about 0.049 mM to about 4.9 mM L (+)-glutamic acid monosodium salt or mixtures thereof and about 1.0 g / and from 1 to about 10.0 g / l HA. In another example, from about 1.0 g / l to about 10.0 g / l of HA is replaced with about 50 g / l of soy peptone. In another example, a 10 mM phosphate buffer solution having a pH of about 6.5 to about 7.8 may contain about 0.25 mM to about 12.5 mM HEPES, about 0.01 mM to about 0.5 mM magnesium chloride, about 0.01 mM to about 0.5 mM calcium chloride, about 50 g / L sucrose, from about 0.049 mM to about 2.45 mM L (+)-glutamic acid or from about 0.049 mM to about 2.45 mM L (+)-glutamic acid monosodium salt or mixtures thereof, from about 1.0 g / L to About 10.0 g / l HA and about 50 g / l Soy peptone.

In one example, the storage temperature of the small dose stable viral composition is about 5 ° C. In another particular embodiment, the viral composition exhibits a PFU reduction of less than about 1.0 log PFU after one year of storage at about 1 ° C. to about 10 ° C. In another embodiment, the viral composition exhibits at least about 4.0 log PFU per 0.2 ml after 1 year of storage at about 1 ° C. to about 10 ° C.

In one example, lyophilization of the viral composition comprises: (a) adding about 0.5 ml to 0.6 ml of the viral composition into a vial to cool to a temperature of about 5 ° C .; (b) placing the vial on a lyophilization shelf to reduce the shelf temperature from 5 ° C. to −50 ° C. at a rate of about −1.0 ° C. per minute to about −2.0 ° C. per minute; (c) the shelf temperature is maintained at about −50 ° C. for 60 minutes; (d) reducing the lyophilization chamber pressure to 0.10 Torr and maintaining the shelf temperature at about −50 ° C. for 30-60 minutes; (e) increasing the shelf temperature from −50 ° C. to 0 ° C. at a rate of about 1.0 ° C. to about 2.0 ° C. at about 0.10 Torr and maintaining the shelf temperature at about 0 ° C. for about 540 minutes to about 720 minutes; (f) increasing the shelf temperature from 0 ° C. to 15 ° C. at a rate of about 0.5 ° C. per minute at about 0.10 Torr and maintaining the shelf temperature at about 15 ° C. for about 600 minutes to about 720 minutes; (g) further limited to filling the vial with nitrogen gas and hermetically sealing it.

In another embodiment, lyophilization of the viral composition comprises (a) adding about 0.5 ml to 0.6 ml of the viral composition into a vial to cool to a temperature of about 5 ° C .; (b) freezing the lyophilized shelf to a temperature of about −70 ° C .; (c) placing the vial on a lyophilized shelf to maintain the temperature at about −70 ° C. for about 60 minutes; (d) reducing the lyophilization chamber pressure to 0.10 Torr and increasing the shelf temperature from about −70 ° C. to −50 ° C. at a rate of about 1.0 ° C. per minute; (e) increasing the shelf temperature from −50 ° C. to 0 ° C. at a rate of about 1.0 ° C. to about 2.0 ° C. at about 0.10 Torr and maintaining the shelf temperature at about 0 ° C. for about 540 minutes to about 720 minutes; (f) increasing the shelf temperature from 0 ° C. to 15 ° C. at a rate of about 0.5 ° C. per minute at about 0.10 Torr and maintaining the shelf temperature at about 15 ° C. for about 600 minutes to about 720 minutes; (g) further limited to filling the vial with nitrogen gas to hermetically seal it.

In another embodiment, the present invention relates to a method for preparing a large amount (mass) of lyophilized stable virus composition. In certain embodiments, the present invention provides a method for preparing a lyophilized tray comprising (a) placing a liquid virus composition of at least 50 ml in a lyophilized tray; (b) the virus composition is frozen in a liquid nitrogen bath for at least 20 minutes; (c) lyophilizing the viral composition, wherein the lyophilized viral composition is characterized by a PFU reduction of less than about 0.5 log PFU relative to the viral composition prior to lyophilization. (Or a large amount) of a lyophilized stable virus composition. In another embodiment, the high volume viral composition exhibits at least 5.0 log PFU per dose after lyophilization. In one example, the glass transition temperature is about -35 ° C. In another example, the glass transition temperature is about -30 ° C to about -40 ° C. In another example, the lyophilized tray is a Lyoguard ^® lyophilized tray (WL Gore and Associates; Newark, DE). In one example, the high volume viral composition is at least 500 ml per lyophilized tray. In another embodiment, the high volume viral composition is at least 1000 ml per lyophilized tray. In one example, RSV is defined as RSV-A, RSV-B or chimeric recombinant RSV (RSV-AB) comprising one or more antigens of each of Groups A and B, where PIV is PIV-1, PIV-2 or PIV- It is further limited to three.

In one example, the high volume viral composition comprises sodium and / or potassium monobasic and dibasic salts and is formulated in a 5.0 mM to about 20 mM phosphate buffer solution having a pH of about 6.5 to about 7.8. In another example, the 5.0 mM to about 20 mM phosphate buffer solution further comprises about 2.5 mM to about 25 mM HEPES. In another specific example, the 5.0 mM to about 20 mM phosphate buffer solution further comprises about 0.1 mM to about 1 mM magnesium chloride and about 0.1 mM to about 1 mM calcium chloride.

In certain embodiments, the high volume viral composition comprises sodium and / or potassium mono and dibasic salts and is formulated in a 10 mM phosphate buffer solution having a pH of about 6.5 to about 7.8. In another example, the 10 mM phosphate buffer solution further comprises about 2.5 mM to about 25 mM HEPES. In another specific example, the 10 mM phosphate buffer solution further comprises about 0.1 mM to about 1 mM magnesium chloride and about 0.1 mM to about 1 mM calcium chloride.

In one example, 5.0 mM to about 20 mM having a pH of about 6.5 to about 7.8 and comprising about 2.5 mM to about 25 mM HEPES, about 0.1 mM to about 1 mM magnesium chloride, and about 0.1 mM to about 1 mM calcium chloride. Phosphate buffer solutions include sucrose, L (+)-glutamic acid, L (+)-glutamic acid monosodium salt, a mixture of L (+)-glutamic acid and L (+)-glutamic acid monosodium salt, human albumin (HA) and / or Further comprises soy peptone. In another embodiment, 5.0 mM to about 20 mM, having a pH of about 6.5 to about 7.8 and comprising about 2.5 mM to about 25 mM HEPES, about 0.1 mM to about 1 mM magnesium chloride, and about 0.1 mM to about 1 mM calcium chloride. The phosphate buffer solution comprises about 50 g / l sucrose, about 0.049 mM to about 4.9 mM L (+)-glutamic acid or about 0.049 mM to about 4.9 mM L (+)-glutamic acid monosodium salt or mixtures thereof and And from about 1.0 g / l to about 10.0 g / l HA. In one example, about 1.0 g / l to about 10.0 g / l of HA is replaced with about 50 g / l of soy peptone. In another example, a 5.0 mM to about 20 mM phosphate buffer solution having a pH of about 6.5 to about 7.8 may contain about 2.5 mM to about 25 mM HEPES, about 0.1 mM to about 1 mM magnesium chloride, and about 0.1 mM to about 1 mM calcium chloride. , About 50 g / L sucrose, about 0.049 mM to about 4.9 mM L (+)-glutamic acid or about 0.049 mM to about 4.9 mM L (+)-glutamic acid monosodium salt or mixtures thereof, about 1.0 g / l to about 10.0 g / l HA and about 50 g / l Soy peptone.

In another embodiment, 10 mM phosphate having a pH of about 6.5 to about 7.8 and comprising about 2.5 mM to about 12.5 mM HEPES, about 0.1 mM to about 0.5 mM magnesium chloride, and about 0.1 mM to about 0.5 mM calcium chloride. The buffer solution is about 50 g / l sucrose, about 0.049 mM to 2.45 mM L (+)-glutamic acid or about 0.049 mM to about 2.45 mM L (+)-glutamic acid monosodium salt or mixtures thereof and about 1.0 g / l to about 10.0 g / l HA further. In another embodiment, about 1.0 g / l to about 10.0 g / l of HA is replaced with about 50 g / l of soy peptone. In another embodiment, a 10 mM phosphate buffer solution having a pH of about 6.5 to about 7.8 comprises about 2.5 mM to about 12.5 mM HEPES, about 0.1 mM to about 0.5 mM magnesium chloride, about 0.1 mM to about 0.5 mM calcium chloride, About 50 g / l sucrose, about 0.049 mM to 2.45 mM L (+)-glutamic acid or about 0.049 mM to about 2.45 mM L (+)-glutamic acid monosodium salt or mixture thereof, about 1.0 g / l To about 10.0 g / l HA and about 50 g / l Soy peptone.

In another particular example, lyophilization of a large volume viral composition comprises (a) placing a lyophilized tray containing the frozen viral composition at a temperature of about -50 ° C on a lyophilized shelf precooled to a temperature of about -50 ° C. Hold for about 60 minutes; (b) reducing the chamber pressure to 0.10 Torr and increasing the shelf temperature from −50 ° C. to −23 ° C. at a rate of about 0.23 ° C. per minute at about 0.10 Torr; (c) the shelf temperature is maintained at about −23 ° C. for about 80 hours to about 100 hours; (d) reduce the lyophilization chamber pressure to 0.02 Torr and increase the shelf temperature from −23 ° C. to 15 ° C. at a rate of about 0.23 ° C. per minute; (e) the shelf temperature is maintained at about 0.02 Torr at about 15 ° C. for about 30 to about 40 hours; (f) increasing the shelf temperature from 15 ° C. to 25 ° C. at a rate of about 0.17 ° C. per minute at 0.02 Torr; (g) the shelf temperature is maintained at about 25 ° C. at about 0.02 Torr for about 10 hours; (h) filling the chamber with nitrogen gas and hermetically sealing the tray in an aluminum pouch under nitrogen gas.

In another particular example, lyophilization of a large volume viral composition comprises (a) placing a lyophilized tray containing the frozen viral composition at a temperature of about −70 ° C. on a lyophilized shelf precooled to a temperature of about −70 ° C. Hold for about 60 minutes; (b) reducing the chamber pressure to 0.10 Torr and increasing the shelf temperature from −70 ° C. to −23 ° C. at a rate of about 0.23 ° C. per minute; (c) the shelf temperature is maintained at about 0.10 Torr at about -23 ° C. for about 80 hours to about 100 hours; (d) reduce the chamber pressure to 0.02 Torr and increase the shelf temperature from −23 ° C. to 15 ° C. at a rate of about 0.23 ° C. per minute; (e) the temperature was maintained at 0.02 Torr at about 15 ° C. for about 30 to 40 hours; (f) increasing the shelf temperature from 15 ° C. to 25 ° C. at a rate of about 0.17 ° C. per minute at 0.02 Torr; (g) the temperature is maintained at about 25 ° C. for about 10 hours; (h) filling the chamber with nitrogen gas and hermetically sealing the tray in an aluminum pouch under nitrogen gas.

In another embodiment, the present invention relates to a method of preparing a storage stable frozen liquid virus composition. In certain embodiments, the present invention provides a method for preparing a metal plate comprising (a) equilibrating a metal plate in a liquid nitrogen bath; (b) placing the liquid viral composition in a suitable container means; (c) inserting the container of step (b) into a metal holder; (d) leaving the metal holder on the equilibrated metal plate of step (a) for about 10 minutes; (e) removing the container from the metal holder; (f) storing the container at a temperature of about -20 ° C to about -70 ° C, wherein after steps (a) to (f) the viral composition is characterized by a PFU reduction of less than about 0.5 log PFU after storage for 6 months The present invention relates to a method for preparing a storage stable frozen liquid virus composition comprising RSV, PIV or a combination thereof. In certain embodiments, the container means is an intranasal spray device. In one example, the intranasal spray device is a BD Accuspray ^™ intranasal spray device (BD Medical Pharmaceutical Systems; Franklin Lakes, NJ). In another embodiment, the metal holder is aluminum. In another embodiment, the metal holder is stainless steel. In another embodiment, the viral composition exhibits at least 4.0 log PFU / 0.2 ml after steps (a) to (f). In another embodiment, the viral composition exhibits at least 4.0 log PFU / 0.2 ml after 6 months storage at a temperature of -20 ° C. In another embodiment, the viral composition exhibits at least 4.0 log PFU / 0.2 ml after 6 months storage at a temperature of −70 ° C. In certain embodiments, the liquid viral composition is formulated in the absence of protein stabilizers. In one example, RSV is defined as RSV-A, RSV-B or chimeric recombinant RSV (RSV-AB) comprising one or more antigens of each of Groups A and B, where PIV is PIV-1, PIV-2 or PIV- It is further limited to three.

In another particular embodiment, the liquid viral composition is formulated in a 5.0 mM to about 20 mM phosphate buffer solution containing sodium and / or potassium monobasic and dibasic salts and having a pH of about 6.5 to about 7.8. In another example, the 5.0 mM to about 20 mM phosphate buffer solution further comprises about 0.25 mM to about 25 mM HEPES. In another specific example, the 5.0 mM to about 20 mM phosphate buffer solution further comprises about 0.01 mM to about 1 mM magnesium chloride and about 0.01 mM to about 1 mM calcium chloride.

In certain embodiments, the liquid viral composition comprises sodium and / or potassium mono and dibasic salts and is formulated in a 10 mM phosphate buffer solution having a pH of about 6.5 to about 7.8. In another example, the 10 mM phosphate buffer solution further comprises about 0.25 mM to about 25 mM of HEPES. In another specific example, the 10 mM phosphate buffer solution further comprises about 0.01 mM to about 1 mM magnesium chloride and about 0.01 mM to about 1 mM calcium chloride.

In one example, 5.0 mM to about 20 mM having a pH of about 6.5 to about 7.8 and comprising about 0.25 mM to about 25 mM HEPES, about 0.01 mM to about 1 mM magnesium chloride, and about 0.01 mM to about 1 mM calcium chloride. The phosphate buffer solution further comprises sucrose and L (+)-glutamic acid, L (+)-glutamic acid monosodium salt or mixtures thereof. In another embodiment, from 5.0 mM to about 20 mM having a pH of about 6.5 to about 7.8 and comprising about 0.25 mM to about 25 mM HEPES, about 0.01 mM to about 1 mM magnesium chloride and about 0.01 mM to about 1 mM calcium chloride The phosphate buffer solution is about 75 g / l sucrose and about 4.9 mM L (+)-glutamic acid or about 4.9 mM L (+)-glutamic acid monosodium salt or about 4.9 mM L (+)-glutamic acid and L Further comprises a mixture of (+)-glutamic acid monosodium salt.

In another embodiment, 10 mM phosphate having a pH of about 6.5 to about 7.8 and comprising about 0.25 mM to about 25 mM HEPES, about 0.01 mM to about 1 mM magnesium chloride, and about 0.01 mM to about 1 mM calcium chloride The buffer solution is about 75 g / l sucrose and about 4.9 mM L (+)-glutamic acid or about 4.9 mM L (+)-glutamic acid monosodium salt or about 4.9 mM L (+)-glutamic acid and L ( +)-Glutamic acid monosodium salt further.

In another embodiment, the present invention provides at least a storage temperature of about 1 ° C. to about 10 ° C., prepared according to the method of freezing the virus composition below its glass transition temperature for up to about 60 minutes and lyophilizing the virus composition. A lyophilized small dose storage stable virus composition that is stable for one year.

In another embodiment, the present invention is directed to placing a liquid virus composition of at least 50 ml in a lyophilized tray; The virus composition is frozen in a liquid nitrogen bath below its glass transition temperature for at least about 20 minutes; A lyophilized bulk storage stable virus composition exhibiting a PFU reduction of less than about 5.0 log PFU relative to the pre-lyophilized viral composition, prepared according to the method of lyophilizing the viral composition.

In another embodiment, the present invention provides a process for the preparation of (a) equilibrating a metal plate in a liquid nitrogen bath; (b) placing the liquid viral composition in a suitable container means; (c) inserting the container of step (b) into a metal holder; (d) leaving the metal holder on the equilibrated metal plate of step (a) for about 10 minutes; (e) removing the container from the metal holder; (f) The viral composition following steps (a) to (f), prepared according to the method of storing the container at a temperature of about -20 ° C to about -70 ° C, has a PFU reduction of less than about 0.5 log PFU after storage for 6 months. A storage stability freeze liquid virus composition is shown.

In another specific embodiment, the present invention relates to an immunogenic composition comprising a viral composition prepared according to the lyophilization method of the present invention, wherein the virus is dissolved, diluted or suspended in a pharmaceutically acceptable carrier.

In another embodiment, the present invention relates to an immunogenic composition comprising a frozen liquid virus composition prepared according to the method of the present invention.

Other features and advantages of the invention will be apparent from the following detailed description, preferred embodiments thereof, and the claims.

1 is a schematic of a BD Accuspray ^™ device in a 96-well aluminum holder used to freeze the formulation, indicated by the following symbols: (1) aluminum or steel holder, (2) formulation filled in the apparatus, (3 ) BD Accuspray device, (4) stopper and (5) empty well.

FIG. 2 shows the frozen kinetic of the -70 ° C. freezer formulation as compared to freezing the same formulation with liquid nitrogen. After introducing the liquid formulation into the BD Accuspray ^™ apparatus, freezing was performed by placing the aluminum holder on a metal surface cooled with liquid nitrogen, or by placing the aluminum holder in a -70 ° C. freezer.

Hereinafter, the present invention will be described with respect to a method for preparing a storage stable virus composition facing the art. In certain embodiments, the invention described hereinafter is directed to the respiratory syncytial virus (RSV) facing the art for use in immunogenic compositions that prevent or ameliorate respiratory diseases in infants, children, the elderly, and those with compromised immunity. The methods of preparing storage stable viral compositions, including parainfluenza virus (PIV) or combinations thereof, will be discussed.

In another specific embodiment, the present invention is directed to simple herpes virus, cytomegalovirus, Epstein-Barr virus, as required in the art for use in immunogenic compositions that prevent or ameliorate diseases caused by one or more of the following viruses: Varicella zoster virus, Mumps virus, measles virus, influenza virus, polio virus, rhinovirus, adenovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, norwalk virus, toga virus, alpha virus, Storage stable viruses, including one or more viruses, such as rubella virus, rabies virus, marburg virus, ebola virus, papilloma virus, polyoma virus, metapneumovirus, corona virus, bullous stomatitis virus, venezuelan equine encephalitis virus, etc. The discussion focused on a method of manufacturing the sacred.

Thus, in certain embodiments, the present invention relates to a method of making a lyophilized small dose viral composition. In certain embodiments, the present invention relates to a method of making a lyophilized small dose viral composition that is storage stable for at least one year at a storage temperature of about 1 ° C to about 10 ° C. In another particular embodiment, the present invention is directed to a method of making a lyophilized large (or large) virus composition. In certain embodiments, the present invention relates to a process for the preparation of large (or large) lyophilized viral compositions that exhibit a reduction of less than about 0.5 log PFU (plaque forming units) relative to the composition prior to lyophilization. In another embodiment, the present invention relates to a method of preparing a frozen liquid virus composition. In certain embodiments, the invention relates to a method of making a frozen liquid virus composition exhibiting a reduction of less than about 0.5 log PFU after storage for 6 months. In another embodiment, the present invention provides storage stable viral compositions prepared according to the methods of the present invention. In another embodiment, an immunogenic composition prepared according to the method of the present invention is provided.

A. Viral Composition

RSV belongs to the genus Pneumoviridae , which is classified as a paramyxovirus family. The virion contains 15,222 base pairs of single stranded negative sense RNA encoding 10 viral proteins. These ten proteins include three envelope-bound glycoproteins named G, F and SH; Two matrix proteins M and M2, three nucleocapsid proteins L, N and P, and nonstructural proteins 1B and 1C.

Two groups of RSV, group A and group B, were identified based on antigenic differences in G protein and, to a lesser extent, F protein. Antigen differences can be identified within the two groups. G proteins exhibit a high degree of variability with up to 20% difference in G protein sequence in RSV group A and only 53% amino acid homology between RSV groups A and B. Hereinafter, "RSV group A" will be referred to as "RSV-A", "RSV group B" will be referred to as "RSV-B".

Storage stable RSV compositions (or RSV / PIV complexes) prepared according to the methods of the present invention include cp RSV mutants ( cp RSV), temperature sensitive RSV mutants ( ts RSV), cp temperature sensitive RSV mutants ( cpts RSV), Any attenuated RSV (eg, attenuated RSV-A and attenuated RSV-B), including, but not limited to, cold adapted RSV mutants ( ca RSV), small plaque RSV mutants ( sp RSV), and the like. . For example, US Pat. Nos. 5,882,651, 5,932,222, 5,993,824, 6,077,514 and 6,284,254, each of which are incorporated herein by reference, disclose methods of making various attenuated RSV phenotypes. In a preferred embodiment, the attenuated RSV of the present invention is all recombinant variants made from this strain, including the cpts RSV 248/404 (ATCC VR2452) (also referred to as LRSV-404) and recombinant RSV-AB strains. Other exemplary RSV strains of the invention include (a) rA2cp248 / 404ΔSH (aka LRSV-rA36); (b) rA2cp248 / 404 / 1030ΔSH (aka LRSV-rA38); (c) rA2cp248 / 404/1030 (aka LRSV-rA39); (d) rA2cp248 / 404ΔNS2 (aka LRSV-rA41); (e) rABcp248 / 404/1030 (aka LRSV-rAB1); (f) rABcp248 / 404ΔSH (aka LRSV-rAB2); (g) rABcp248 / 404ΔNS2 (aka LRSV-rAB4); (h) cpts RSV530 / 1009 (ATCC VR2451) and rA2cp530 / 1009ΔNS2 (aka LRSV-rA42); rA2cp530 / 1009/404 (aka LRSV-rA43); All recombinant variants made from this strain are included, including recombinant RSV-AB strains such as rABcp530 / 1009ΔNS2 (aka LRSV-rAB3) and rABcp530 / 1009/404 (aka LRSV-rAB6).

Human parainfluenza virus type 3 (PIV-3) is a member of the recently named Respirovirus genus of the paramyxovirus family. Its genome is a single stranded negative-sense RNA of 15,462 nucleotides in length. At least eight proteins are encoded by PIV-3: nucleocapsid protein NP, phosphoprotein P, nonstructural protein C, D protein, matrix protein M, fusion glycoprotein F, hemagglutinin-neurami Nidase Protein HN and Large Polymerase Proteins L. HN and F proteins are envelope bound surface glycoproteins that are major neutralizing and protective antigens. Significant sequence differences between comparable PIV HN or F proteins among the PIV types (eg types 1, 2 and 3) are believed to be the basis for the type specificity of protective immunity.

Human parainfluenza virus type 1 (PIV-1) is another member of the genus Respirovirus of paramyxoviruses. Its genome is a single stranded negative-sense RNA that is approximately 15,600 nucleotides in length. The gene product groups encoded by PIV-1 include nucleocapsid protein NP, phosphoprotein P (and a number of other gene products encoded by P ORF), matrix protein M, fusion glycoprotein F, H. Magglutinin-neuraminidase protein HN and large polymerase protein L.

Human parainfluenza virus type 2 (PIV-2) is a member of the genus Rubulavirus of paramyxoviruses. Its genome is a single stranded negative-sense RNA that is approximately 15,654 nucleotides in length. The gene product groups encoded by PIV-2 include nucleocapsid protein NP, phosphoprotein P, V protein, matrix protein M, fusion glycoprotein F, hemagglutinin-neuramidase protein HN and large polymerase protein L. It includes.

Storage stable PIV compositions (or RSV / PIV complexes) prepared according to one of the methods of the present invention include cp PIV mutants ( cp PIV), temperature-sensitive PIV mutants ( ts PIV), cp temperature-sensitive PIV mutants (a cpts PIV), a cold adapted PIV mutant ( ca PIV), a small plaque PIV mutant ( sp PIV), and the like, and any attenuated PIV. In a preferred embodiment, the attenuated PIV of the invention is a cp PIV-3 mutant of cp-45 (or JS cp45) designated as a JS wild type strain. In a preferred embodiment, the PIV-3 cp-45 mutations are further attenuated using the "menu" of the attenuated PIV-3 mutations disclosed in US Pat. Nos. 6,410,023 and 5,869,036, each of which are incorporated by reference.

In another embodiment, the storage stable viral composition prepared according to one of the methods of the present invention comprises one or more viruses or vectors thereof illustrated in Table 1 below, but not limited thereto.

Virus family I. piconavirus Enterovirus Polio virus Coxsackie virus Echo virus Reno virus Hepatitis A Virus II. Calicivirus Norwalk Group Virus III. Togaviruses and Flaviviruses Toga virus (such as dengue virus) Alpha virus Flavi virus (for example, hepatitis C virus) Rubella virus IV. Coronavirus Corona virus V. Rhabdovirus Rabies virus VI. Filovirus Marburg virus Ebola virus VII. Paramyxoviruses Parainfluenza virus Mumps virus Measles virus Respiratory syncytial virus Metapneumovirus VIII. Orthomyxoviruses Orthomyxoviruses (such as influenza viruses) IX. Bunya virus Buniya virus X. Arena Virus Arena virus XI. Leo Virus Leo virus Rota virus Orbi virus XII. Retrovirus

Virus family Human T Cell Leukemia Virus Type I Human T Cell Leukemia Virus Type II Human immunodeficiency virus
(E.g. Type I and Type II) Apes Immunodeficiency Virus Lenti virus XIII. Paphovirus Polyoma virus Papilloma virus XIV. Parvovirus Paribovirus HV. Herpes virus Simple Herpes Virus Epstein-Barr Virus Cytomegalovirus Varicella zoster virus Human Herpes Virus-6 Human Herpes Virus-7 Sercophythecin Herpes Virus 1 (B Virus 1) XVI. Poxvirus Fox virus XVIII. Hepadnavirus Hepatitis B Virus XIX. Adenovirus

B. Small Storage Stability Virus

In certain embodiments, the present invention relates to a method of making a small dose storage stable virus composition. In one embodiment, the present invention relates to a method of making a small dose storage stable viral composition comprising RSV, PIV or a combination thereof. The method includes freezing the viral composition below its glass transition temperature (T _g ) for up to about 60 minutes and lyophilizing the viral composition. The lyophilized virus composition, in the form of a solid powder or cake, is stable for at least one year at a storage temperature of about 1 ° C to about 10 ° C. Lyophilized small dose storage stable viral compositions are particularly useful as single or multiple dose immunogenic compositions wherein the lyophilized powder is stored for a given amount of time.

A “small dose” viral composition is about 100 μl to about 5 ml. In certain embodiments, the small dose viral composition is about 200 μl to about 1 ml. In one example, the dose of viral composition is 500 μl.

In certain embodiments, the small dose viral composition is frozen and lyophilized in a suitable container means. Typically, suitable container means for small dose viral compositions are containers that can withstand freezing and lyophilization temperatures and vacuum pressures. For example, container means suitable for preparing small dose storage stability compositions are vials, tubes, syringes, two-stage syringes or intranasal spray devices. Reference: US Pat. Nos. 5,489,266, 5,732,837 and 4,084,330, each of which is incorporated herein by reference. Additional lyophilized container means are known and readily available to those skilled in the art.

1. Small Dose Viral Formulations

The "RSV composition", "PIV composition" or "RSV / PIV composition" as defined hereinafter is a virus (ie, RSV, PIV or RSV / PIV), typically about 10 ³ to 10 ⁷ PFU of attenuated virus and medicament per ml Academically acceptable carriers. Pharmaceutically acceptable carriers include buffers, saline, water, water for injection (WFI), protein stabilizers, sugars, amino acids, antifreeze agents and the like.

Small dose viral compositions comprise sodium and / or potassium monobasic and dibasic salts and are formulated in a 5.0 mM to about 20 mM phosphate buffer solution having a pH of about 6.5 to about 7.8. In certain formulations, the 5.0 mM to about 20 mM phosphate buffer solution further comprises about 0.25 mM to about 25 mM HEPES, about 0.01 mM to about 1 mM magnesium chloride, and about 0.01 mM to about 1 mM calcium chloride.

In certain formulations, from 5.0 mM to about 20 mM phosphate buffer (pH from about 6.5 to about 25 mM) comprising from about 0.25 mM to about 25 mM of HEPES, from about 0.01 mM to about 1 mM of magnesium chloride, from about 0.01 mM to about 1 mM of calcium chloride. The small dose viral composition formulated in about 7.8) further comprises sucrose, L (+)-glutamic acid, L (+)-glutamic acid monosodium salt or mixtures thereof, human albumin (HA) and / or soy peptone. In another particular formulation, a 10 mM phosphate buffer solution (pH about 6.5 to about 7.8) comprising about 0.25 mM to about 12.5 mM HEPES, about 0.01 mM to about 0.5 mM magnesium chloride, and about 0.01 mM to about 0.5 mM calcium chloride. ) Is about 0.049 mM to about 2.45 mM L (+)-glutamic acid or about 0.049 mM to about 2.45 mM L (+)-glutamic acid monosodium salt or mixtures thereof, about 50 g / L sucrose and about 1.0 g / l to about 10.0 g / l HA further. In another particular formulation, about 1.0 g / l to 10.0 g / l of HA is replaced with about 50 g / l of soy peptone (also known as Hy-Soy ^® ; Quest International; Chicago, IL). In another formulation, a stable, low dose viral composition comprises about 0.25 mM to about 25 mM HEPES, about 0.01 mM to about 1 mM magnesium chloride, about 0.01 mM to about 1 mM calcium chloride, about 50 g / L sucrose, about 0.049 mM to about 4.9 mM L (+)-glutamic acid or about 0.049 mM to about 4.9 mM L (+)-glutamic acid monosodium salt or mixtures thereof, about 1.0 g / l to about 10.0 g / l HA and Formulated in 5.0 mM to about 20 mM phosphate buffer solution (pH about 6.5 to about 7.8) comprising about 50 g / l Soy peptone.

2. Freeze Rate and Lyophilization of Small Dose Viruses

As described above, the method of making a small dose storage stable virus composition comprises (a) freezing the virus composition below its glass transition temperature within a time of about 60 minutes or less, and (b) lyophilizing the virus composition, The lyophilized virus composition is stable for at least one year at a storage temperature of about 1 ° C to about 10 ° C.

The T _g of the viral composition is typically about -35 ° C. The T _g of the viral composition is less than about −35 ° C. (eg about −42 ° C.) in the presence of a “carry over” salt such as sodium chloride. For example, sodium chloride is a component of viral growth medium but is not a component of small dose formulations. Thus, certain viral preparations will contain a residual amount (ie, "carry over") of sodium chloride, and therefore T _g may be less than about -35 ° C, but typically not lower than about -50 ° C.

The term "glass transition temperature" or "T _g " refers to approximately the midpoint of the temperature range in which the transition from liquid to glass state occurs. The rate at which the viral composition reaches its T _g is important for virus stability during lyophilization (see Example 2) and long term virus storage stability (see Example 3). In other words, the faster the freezing rate, the more stable the virus composition is produced, thereby reducing the loss of efficacy of the viral composition.

The term "freeze rate" means the rate at which the viral composition reaches its T _g . The freezing rate can be calculated as an approximation rate of the temperature decrease during freezing. For example, if the initial temperature of the virus composition is a 5 ℃, if frozen to -35 ℃ T _g thereof in 40 minutes, "freezing rate" is -1 ℃ / min. Under similar conditions, the frozen kinetic may vary from individual container or, in large quantities, may exhibit deviations at different points. Thus, the freezing rate is the average freezing rate measured at different locations of the material loaded in the tray or observed in the container.

The freezing rate of the low dose viral composition is from about -0.5 ° C / min to about -2.5 ° C / min. In one example, T _g reaches a time of 60 minutes or less. In another embodiment, T _g reaches a time of 40 minutes or less. In another embodiment, T _g reaches 20 or less hours. The T _g of the viral composition can be readily determined by one skilled in the art without undue experimentation using thermodynamic measurements such as differential scanning calorimetry (DSC) (Hatley, 1992; Franks, 1992; Carpenter, 2002).

In one example, the lyophilized vial containing the small dose viral composition is precooled to a temperature of about 5 ° C. The vial containing the precooled virus composition is then placed on a lyophilized shelf and frozen at a temperature of at least −50 ° C. at a rate of about −1 ° C./min to about −2 ° C./min. In another embodiment, the vial containing the precooled virus composition is placed directly on a lyophilized shelf pre-frozen to a temperature of -70 ° C.

Lyophilization (or lyophilization) is a dehydration technique in which a sample solution (eg RSV / PIV composition) is frozen and subjected to high vacuum to remove solvent (eg water or buffer) by sublimation. Lyophilization techniques are well known to those skilled in the art (Rey and May, 1999).

In one example, the lyophilized small dose viral composition is prepared as follows: (a) about 0.5 ml to 0.6 ml of the viral composition is introduced into a vial and cooled to a temperature of about 5 ° C .; (b) placing the vial on a lyophilized shelf to reduce the shelf temperature from 5 ° C. to −50 ° C. at a rate of about −1.0 ° C. per minute to about −2.0 ° C. per minute; (c) the shelf temperature is maintained at about −50 ° C. for 60 minutes; (d) the chamber pressure was reduced to 0.10 Torr and the shelf temperature was maintained at about −50 ° C. for 30-60 minutes; (e) increasing the shelf temperature from −50 ° C. to 0 ° C. at a rate of about 1.0 ° C. to about 2.0 ° C. at about 0.10 Torr and maintaining the shelf temperature at about 0 ° C. for about 540 minutes to about 720 minutes; (f) increasing the shelf temperature from 0 ° C. to 15 ° C. at a rate of about 0.5 ° C. per minute at about 0.10 Torr and maintaining the shelf temperature at about 15 ° C. for about 600 minutes to about 720 minutes; (g) further limited to filling the vial with nitrogen gas to hermetically seal it.

In another embodiment, the lyophilized small dose viral composition is prepared as follows: (a) about 0.5 ml to 0.6 ml of the viral composition is introduced into a vial and cooled to a temperature of about 5 ° C .; (b) freezing the lyophilized shelf to a temperature of about −70 ° C .; (c) placing the vial on a lyophilized shelf to maintain the temperature at about −70 ° C. for about 60 minutes; (d) reducing the chamber pressure to 0.10 Torr and increasing the shelf temperature from about −70 ° C. to −50 ° C. at a rate of about 1.0 ° C. per minute; (e) increasing the shelf temperature from −50 ° C. to 0 ° C. at a rate of about 1.0 ° C. to about 2.0 ° C. at about 0.10 Torr and maintaining the shelf temperature at about 0 ° C. for about 540 minutes to about 720 minutes; (f) increasing the shelf temperature from 0 ° C. to 15 ° C. at a rate of about 0.5 ° C. per minute at about 0.10 Torr and maintaining the shelf temperature at about 15 ° C. for about 600 minutes to about 720 minutes; (g) further limited to filling the vial with nitrogen gas to hermetically seal it.

Lyophilized small dose viral compositions (ie, lyophilized cakes) showed a PFU reduction of less than about 1.0 log PFU upon lyophilization, with a decrease of less than about 1.0 log after 1 year storage at about 1 ° C. to about 10 ° C. (See Example 2, Example 3 and Tables 2 and 4-7). In another embodiment, the lyophilized small dose viral composition is at least 4.0 log PFU / 0.2 ml after 1 year storage at about 1 ° C. to about 10 ° C.

C. Large Capacity Lyophilized Stability Virus Compositions

In another embodiment, the present invention relates to a method for preparing a large (or large) lyophilized stable virus composition. In one example, the present invention relates to a method for the preparation of a large (or large) lyophilized stable virus composition comprising RSV, PIV or a combination thereof. This method comprises (a) placing a liquid virus composition of at least 50 ml in a lyophilized tray; (b) freezing the viral composition below its T _g for at least 20 minutes in a liquid nitrogen bath; (c) lyophilizing the viral composition. The lyophilized viral composition showed a decrease of less than about 0.5 log PFU for the viral composition prior to lyophilization treatment. Processes for the preparation of large amounts of lyophilized stable virus compositions are particularly useful for large scale production / preparation of such viral compositions.

The "bulk volume" or "large volume" viral composition, as defined below, is about 50 ml to about 2 l per lyophilized tray. In certain embodiments, the large capacity is about 250 mL to about 1 mL per lyophilized tray. In a particular example, the bulk viral composition is about 1 L per lyophilized tray.

1. High Volume Virus Preparation

Large volume viral compositions are formulated with pharmaceutically acceptable carriers including buffers, saline, water, water for injection (WFI), protein stabilizers, sugars, amino acids, antifreeze agents and the like.

In one example, large volume viral compositions are formulated in phosphate buffered solutions comprising sodium and / or potassium monobasic and dibasic salts. The concentration of phosphate buffer is about 5.0 mM to 20 mM and the pH is about 6.5 to about 7.8.

In another embodiment, the 5.0 mM to about 20 mM phosphate buffer solution further comprises about 2.5 mM to about 25 mM HEPES. In another specific embodiment, the 5.0 mM to about 20 mM phosphate buffer solution further comprises about 0.1 mM to about 1 mM magnesium chloride and about 0.1 mM to about 1 mM calcium chloride.

In certain embodiments, the high volume viral composition is formulated in 10 mM phosphate buffer (pH about 6.5 to about 7.8) and further comprises about 2.5 mM to about 12.5 mM HEPES. In another specific example, the 10 mM phosphate buffer solution further comprises about 0.1 mM to about 0.5 mM magnesium chloride and about 0.1 mM to about 0.5 mM calcium chloride.

In one example, 5.0 mM to about 20 mM phosphate buffer solution (pH 6.5 to 7.8, 2.5-25 mM HEPES, 0.1-1.0 mM magnesium chloride, 0.1-1.0 mM calcium chloride) is sucrose, L (+)-glutamic acid. , L (+)-glutamic acid monosodium salt or mixtures thereof, human albumin (HA) and / or soy peptone. In another embodiment, the 10 mM phosphate buffer solution (pH 6.5 to 7.8, 2.5-12.5 mM HEPES, 0.1-0.5 mM magnesium chloride, 0.1-0.5 mM calcium chloride) is about 50 g / L sucrose, about 0.049 add from about 1.0 g / l to about 10.0 g / l of HA to about 2.45 mM L (+)-glutamic acid or about 0.049 mM to about 2.45 mM L (+)-glutamic acid monosodium salt or mixtures thereof It includes. In one example, about 1.0 g / l to about 10.0 g / l of HA is replaced with about 50 g / l of soy peptone. In another embodiment, the 10 mM phosphate buffer solution (pH about 6.5 to about 7.8) comprises about 2.5 mM to about 12.5 mM HEPES, about 0.1 mM to about 0.5 mM magnesium chloride, and about 0.1 mM to about 0.5 mM calcium chloride. , About 50 g / l sucrose, about 0.049 mM to about 2.45 mM L (+)-glutamic acid or about 0.049 mM to about 2.45 mM L (+)-glutamic acid monosodium salt or mixtures thereof, about 1.0 g / l to about 10.0 g / l HA and about 50 g / ml soy peptone further.

2. Freezing Rate and Lyophilization of Large Viruses

A method of making a large volume lyophilized stable virus composition comprises: (a) placing a liquid virus composition of at least 50 ml in a lyophilized tray; (b) freezing the viral composition below its T _g for at least 20 minutes in a liquid nitrogen bath; (c) lyophilizing the viral composition, wherein the lyophilized viral composition exhibits a PFU reduction of less than about 0.5 log PFU for the viral composition prior to lyophilization.

As detailed in section B.2, the rate at which the small dose viral composition reaches its T _g is important for virus storage stability. Likewise, the rate at which large viral compositions reach their T _g is also important for virus storage stability. Thus, an important step in the preparation of high volume viruses is to freeze the virus composition below its glass transition temperature for at least about 40 minutes in a liquid nitrogen bath. Other parameters important for large-capacity rapid freeze rates are heat transfer, composition and structure of the lyophilized tray. For example, a high surface area lyophilized tray can further reduce the time for a large amount of virus composition to reach its T _g . Lyophilized trays are well known in the art and include stainless steel trays, glass trays, aluminum trays, plastic trays and Lyoguard ^® trays. In one example, the lyophilization tray is a Lyoguard ^® lyophilization tray. Trays are specially formulated to provide excellent heat transfer for high volume lyophilization. The tray consists of a microporous membrane designed to allow mass transfer of water vapor while preventing solid particles from flowing out of the tray during the lyophilization cycle.

The T _g of the viral composition is at a temperature of about -35 ° C. As mentioned above, the residual (or “carry over”) sodium chloride from the virus propagation medium may further reduce T _g , but not below −50 ° C.

In another particular example, lyophilization of the viral composition comprises (a) placing a lyophilized tray comprising the virus composition frozen at a temperature of about −50 ° C. on a lyophilized shelf precooled to a temperature of about −50 ° C. Hold for 60 minutes; (b) reducing the chamber pressure to 0.10 Torr and increasing the shelf temperature from −50 ° C. to −23 ° C. at a rate of about 0.23 ° C. per minute at 0.1 Torr; (c) the shelf temperature is maintained at about −23 ° C. for about 80 hours to about 100 hours; (d) reduce the chamber pressure to 0.02 Torr and increase the shelf temperature from −23 ° C. to 15 ° C. at a rate of about 0.23 ° C. per minute; (e) the shelf temperature is maintained at about 0.02 Torr at about 15 ° C. for about 30 to about 40 hours; (f) increasing the shelf temperature from 15 ° C. to 25 ° C. at a rate of about 0.17 ° C. per minute at 0.02 Torr; (g) the shelf temperature is maintained at about 25 ° C. at about 0.02 Torr for about 10 hours; (h) filling the chamber with nitrogen gas and hermetically sealing the tray in an aluminum pouch under nitrogen gas.

In another particular example, lyophilization of a large volume viral composition comprises (a) placing a lyophilized tray containing the frozen viral composition at a temperature of about −70 ° C. on a lyophilized shelf precooled to a temperature of about −70 ° C. Hold for about 60 minutes; (b) reducing the chamber pressure to 0.10 Torr and increasing the shelf temperature from −70 ° C. to −23 ° C. at a rate of about 0.23 ° C. per minute; (c) the shelf temperature is maintained at about 0.10 Torr at about -23 ° C. for about 80 to 100 hours; (d) reduce the chamber pressure to 0.02 Torr and increase the shelf temperature from −23 ° C. to 15 ° C. at a rate of about 0.23 ° C. per minute; (e) the temperature is maintained at 0.02 Torr at about 15 ° C. for about 30 to about 40 hours; (f) increasing the shelf temperature from 15 ° C. to 25 ° C. at a rate of about 0.17 ° C. per minute at 0.02 Torr; (g) the shelf temperature is maintained at about 25 ° C. for about 10 hours; (h) filling the chamber with nitrogen gas and hermetically sealing the tray in an aluminum pouch under nitrogen gas.

Lyophilized bulk virus compositions (ie, lyophilized cakes) showed a PFU reduction of less than about 1.0 log PFU upon lyophilization, and less than about 1.0 log PFU of PFU after one year storage at about 1 ° C. to about 10 ° C. A decrease was shown (see Example 4).

D. Liquid Viral Compositions

In another embodiment, the present invention relates to a method of preparing a storage stable liquid viral composition. In one example, the present invention relates to a method of preparing a storage stable liquid viral composition comprising RSV, PIV or a combination thereof. This method comprises (a) equilibrating the metal plate in a liquid nitrogen bath; (b) placing the liquid viral composition in a suitable container means; (c) inserting the container of step (b) into a metal holder; (d) leaving the metal holder on the equilibrated metal plate of step (a) for about 10 minutes; (e) removing the container from the metal holder; (f) storing the container at a temperature of about -20 ° C to about -70 ° C.

1. Freezing and thawing liquid viruses

As mentioned in step (f), the container containing the frozen virus composition is stored at about −20 ° C. to about −70 ° C. Thawing of the viral composition at room temperature brings the virus composition back to the liquid state, and the thawed liquid viral composition shows a PFU reduction of less than about 0.5 log PFU after storage for 6 months. In one example, the thawed liquid virus composition is at least 4.0 log PFU / 0.2 ml. In another embodiment, the thawed liquid virus composition is at least 4.0 log PFU / 0.2 ml after storage at −20 ° C. for 6 months. In another embodiment, the thawed liquid virus composition is at least 4.0 log PFU / 0.2 ml after storage at −70 ° C. for 6 months.

Typically, container means suitable for liquid viral compositions are containers that are temperature resistant to temperatures ranging from about -20 ° C to about -70 ° C. For example, suitable container means for preparing storage stable liquid compositions are vials, tubes, syringes or intranasal spray devices. In certain instances, the container is an intranasal spray device. In one example, the intranasal spray device is a BD Accuspray ^™ intranasal spray device or similar intranasal spray device available from BD Pharmaceutical Systems (Franklin Lakes, NJ).

The freezing rate of the liquid viral composition is important for virus storage stability (see Example 5). The liquid nitrogen bath is used to rapidly freeze the viral composition. The metal plate of step (a) is any metal that properly transfers heat to the liquid nitrogen bath and removes heat from the container holder of step (c). Similarly, the metal container holder of step (c) is any metal that transfers heat to the metal plate and removes heat from the container containing the virus. In one example, the metal container holder is aluminum. In another example, the metal container holder is stainless steel.

2. Liquid Viral Formulations

Liquid viral compositions are formulated with pharmaceutically acceptable carriers including buffers, saline, water, water for injection (WFI), sugars, amino acids, antifreeze agents and the like.

Liquid viral compositions are formulated in a 5.0 mM to about 20 mM phosphate buffer solution containing sodium and / or potassium monobasic and dibasic salts and having a pH of about 6.5 to about 7.8. In one example, the 5.0 mM to about 20 mM phosphate buffer solution further comprises about 0.25 mM to about 25 mM of HEPES. In another specific example, the 5.0 mM to about 20 mM phosphate buffer solution further comprises about 0.01 mM to about 1 mM magnesium chloride and about 0.01 mM to about 1 mM calcium chloride.

In certain embodiments, the liquid viral composition comprises sodium and / or potassium mono and dibasic salts and is formulated in a 10 mM phosphate buffer solution having a pH of about 6.5 to about 7.8. In another example, the 10 mM phosphate buffer solution further comprises about 0.25 mM to about 25 mM of HEPES. In another specific example, the 10 mM phosphate buffer solution further comprises about 0.01 mM to about 1 mM magnesium chloride and about 0.01 mM to about 1 mM calcium chloride.

In one example, 5.0 mM to about 20 mM having a pH of about 6.5 to about 7.8 and comprising about 0.25 mM to about 25 mM HEPES, about 0.01 mM to about 1 mM magnesium chloride, and about 0.01 mM to about 1 mM calcium chloride. The phosphate buffer solution further comprises sucrose and L (+)-glutamic acid, L (+)-glutamic acid monosodium salt or mixtures thereof and human albumin (HA). In another embodiment, from 5.0 mM to about 20 mM having a pH of about 6.5 to about 7.8 and comprising about 0.25 mM to about 25 mM HEPES, about 0.01 mM to about 1 mM magnesium chloride and about 0.01 mM to about 1 mM calcium chloride The phosphate buffer solution further comprises about 75 g / L sucrose and about 4.9 mM L (+)-glutamic acid or about 4.9 mM L (+)-glutamic acid monosodium salt or mixtures thereof.

In another embodiment, 10 mM phosphate having a pH of about 6.5 to about 7.8 and comprising about 0.25 mM to about 25 mM HEPES, about 0.01 mM to about 1 mM magnesium chloride, and about 0.01 mM to about 1 mM calcium chloride The buffer solution further comprises about 75 g / L sucrose and about 4.9 mM L (+)-glutamic acid or about 4.9 mM L (+)-glutamic acid monosodium salt or mixtures thereof.

Liquid frozen virus compositions (i.e., freezing in spray devices or vials) show a decrease of less than about 0.5 log PFU after "rapid" freezing, and less than about 0.5 log PFU after 6 months storage at about -20 ° C to about -70 ° C. Decreases (see Example 5 and Tables 9-12). In another embodiment, the liquid frozen virus composition is at least 4.0 log PFU / 0.2 ml after storage at about −20 ° C. to about −70 ° C. for 6 months.

E. Immunogenic Virus Compositions

In certain embodiments, the present invention provides immunogenic compositions having a storage stable (freezing) liquid viral composition comprising RSV, PIV, or a combination thereof prepared according to the methods of the present invention. In another embodiment, the present invention is a simple herpes virus, cytomegalovirus, Epstein-Barr virus, varicella zoster virus, Mumps virus, measles virus, influenza virus, polio virus, rhinovirus, adenovirus, hepatitis A virus, type B Hepatitis Virus, Hepatitis C Virus, Norwalk Virus, Toga Virus, Alpha Virus, Rubella Virus, Rabies Virus, Marburg Virus, Ebola Virus, Papilloma Virus, Polyoma Virus, Metapneumovirus, Corona Virus, Bullous Stomatitis Virus Provided is an immunogenic composition having a storage stable (freezing) liquid virus composition, including Venezuelan equine encephalitis virus, and the like.

In certain embodiments, the frozen liquid immunogenic composition is included in an intranasal spray device. Typically, the storage stable (freezing) liquid viral compositions of the invention are formulated and treated to be administered to mammalian subjects using the liquid formulations and processes of the invention (see Section D, Examples 1 and 5) and frozen liquids. And thawed prior to administration to a mammalian subject.

In certain embodiments, the storage stable viral composition of the present invention is a lyophilized solid (or lyophilized cake) composition. In certain embodiments, the lyophilized storage stable viral composition is dissolved, diluted or suspended in a pharmaceutically acceptable carrier to provide an immunogenic composition suitable for mammalian subject (eg, human) administration. Thus, such lyophilized compositions typically include “immunogenic” compositions (eg, attenuated RSV and / or attenuated PIV virus) and “pharmaceutically acceptable carriers”. The term "pharmaceutically acceptable carrier", as used hereinafter, is intended to include any and all solvents known in the art to be suitable for pharmaceutical administration. The use of such media and agents in pharmaceutically active substances is well known in the art. As long as any conventional media or medicament is suitable for the active compound (eg RSV or PIV), such media are used in the compositions of the present invention.

Thus, the immunogenic compositions of the invention are formulated to be suitable for their intended route of administration. Examples of routes of administration include parenteral (eg intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal), mucosal (eg oral, rectal, intranasal, oral, intravaginal, respiratory) and transdermal (topical) administration do. For example, the lyophilized storage stable virus immunogenic composition to be administered by nasal spray comprises one or more of the following components: sterile diluents such as water for injection, saline, buffers (eg acetate, citrate or phosphate) and enteric modulators , For example sodium chloride or dextrose. pH is adjusted with acids or bases, for example hydrochloric acid or sodium hydroxide. Immunogenic compositions are dispensed into spray devices, ampoules, disposable syringes or single / multi dose vials made of glass or plastic.

Parenteral compositions are particularly advantageously formulated in unit dosage forms for dose uniformity and ease of administration. Dosage unit form as used herein means physically discrete units suited as unitary dosage forms for the subject to be treated; Each unit contains a predetermined amount of active compound calculated to provide the desired therapeutic effect with the required pharmaceutical carrier. Specifications for unit dosage forms of the present invention directly depend on and depend upon the inherent properties of the active compound, the particular therapeutic effect desired to be obtained, and the inherent constraints in the combination technology of the active compound for the treatment of the subject.

Pharmaceutically acceptable excipients do not cause side effects, and can, for example, facilitate the administration of the active compound, prolong its life and / or its efficacy in the body, and improve its solubility or preservation thereof in solution. It is to be understood that it means a compound or combination included in a pharmaceutical or immunogenic composition. These pharmaceutically acceptable excipients are well known and can be used by those skilled in the art according to the mode of administration and nature of the active compound selected.

All patents and documents cited herein are hereby incorporated by reference.

F. Examples

Hereinafter, except for the above description, an embodiment performed using conventional standard techniques well known to those skilled in the art will be described. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

Example 1

RSV and PIV Formulation Ingredients

RSV and / or PIV samples described herein were formulated in one of the following phosphate buffers represented by "Formulation A1" through "Formulation E2".

Formulation A1 : 10 mM phosphate buffer comprising 2.5 mM HEPES, 0.1 mM magnesium chloride, 0.1 mM calcium chloride, 0.49 mM L (+)-glutamic acid, 50 g / l sucrose and 1.0 g / l HA (pH 7.0). HA is Grifols ^® 20% (w / v) human albumin (Grifols USA, Los Angeles, CA; Catalog No. 61953-0001-1).

Formulation A2 : 10 mM phosphate buffer comprising 12.5 mM HEPES, 0.5 mM magnesium chloride, 0.5 mM calcium chloride, 2.45 mM L (+)-glutamic acid, 50 g / L sucrose and 1.0 g / L HA (pH 7.0).

Formulation A3 : 10 mM comprising 2.5 mM HEPES, 0.1 mM magnesium chloride, 0.1 mM calcium chloride, 0.49 mM L (+)-glutamic acid, 50 g / L sucrose and 1.0 g / L recombinant HA Phosphate buffer, pH 7.0. Recombinant HA is 20% (w / v) human albumin expressed in yeast cells and is marketed under the trade name of Recombumin ^® (Delta Biotechnology Ltd., Nottingham, United Kingdom).

Formulation A4 : 10 mM comprising 12.5 mM HEPES, 0.5 mM magnesium chloride, 0.5 mM calcium chloride, 2.45 mM L (+)-glutamic acid, 50 g / L sucrose and 1.0 g / L recombinant HA Phosphate buffer, pH 7.0.

Formulation B1 : 10 mM phosphate buffer comprising 2.5 mM HEPES, 0.1 mM magnesium chloride, 0.1 mM calcium chloride, 0.49 mM L (+)-glutamic acid, 50 g / l sucrose and 10 g / l HA (pH 7.0).

Formulation B2 : 10 mM phosphate buffer comprising 12.5 mM HEPES, 0.5 mM magnesium chloride, 0.5 mM calcium chloride, 2.45 mM L (+)-glutamic acid, 50 g / L sucrose and 10 g / L HA (pH 7.0).

Formulation B3 : 10 mM comprising 2.5 mM HEPES, 0.1 mM magnesium chloride, 0.1 mM calcium chloride, 0.49 mM L (+)-glutamic acid, 50 g / l sucrose and 10 g / l recombinant HA Phosphate buffer, pH 7.0.

Formulation B4 : 10 mM comprising 12.5 mM HEPES, 0.5 mM magnesium chloride, 0.1 mM calcium chloride, 2.45 mM L (+)-glutamic acid, 50 g / l sucrose and 10 g / l recombinant HA Phosphate buffer, pH 7.0.

Formulation C1 : 2.5 mM HEPES, 0.1 mM magnesium chloride, 0.1 mM calcium chloride, 0.49 mM L (+)-glutamic acid, 50 g / l sucrose, 50 g / l Soy Peptone (Hy Soy ^® ) and 10 mM phosphate buffer (pH 7.0) containing 1.0 g / l HA.

Formulation C2 : 2.5 mM HEPES, 0.1 mM magnesium chloride, 0.1 mM calcium chloride, 0.49 mM L (+)-glutamic acid, 50 g / l sucrose, 50 g / l soy peptone and about 1.0 g / l 10 mM phosphate buffer (pH 7.0) containing recombinant HA.

Formulation C3 : 2.5 mM HEPES, 0.1 mM magnesium chloride, 0.1 mM calcium chloride, 0.49 mM L (+)-glutamic acid, 50 g / l sucrose, 50 g / l soy peptone and about 1.0 g / l 10 mM phosphate buffer (pH 7.0) containing recombinant HA.

Formulation C4 : 12.5 mM HEPES, 0.5 mM magnesium chloride, 0.1 mM calcium chloride, 2.45 mM L (+)-glutamic acid, 50 g / l sucrose, 50 g / l soy peptone and about 1.0 g / l 10 mM phosphate buffer (pH 7.0) containing HA.

Formulation D1 : 10 mM phosphate comprising 2.5 mM HEPES, 0.1 mM magnesium chloride, 0.1 mM calcium chloride, 0.49 mM L (+)-glutamic acid, 50 g / l sucrose and 50 g / l soy peptone Buffer (pH 7.0).

Formulation D2 : 10 mM phosphate comprising 12.5 mM HEPES, 0.5 mM magnesium chloride, 0.5 mM calcium chloride, 2.45 mM L (+)-glutamic acid, 50 g / l sucrose and 50 g / l soy peptone Buffer (pH 7.0).

Example 2

Effect of Freezing Rate During Lyophilization on the Efficacy of Small Dose RSV and / or PIV Formulations

In this example, the freezing rate of small dose RSV and / or PIV formulations was investigated to determine the optimal freezing conditions necessary to minimize viral efficacy loss.

First, three samples containing LRSV-404, PIV3-cp45 and LRSV-404 / PIV3-cp45 complexes were tested (Table 2). Virus aggregates used in these formulations were prepared as clinical materials for Phase 1 and Phase 2 human clinical trials. Each virus sample was formulated using "Formulation A1" as described in Example 1. Samples were charged to 2 ml vials (0.6 ml per vial), precooled to a temperature of about 5 ° C. and placed on a precooled (-50 ° C.) shelf of the lyophilizer. The glass transition temperature (T _g ) (about −35 ° C. ± 5 ° C.) of the viral composition was reached after about 40 minutes, corresponding to a freezing rate of about −1.0 ° C. per minute. After freezing, it was subjected to a lyophilization cycle comprising first drying at 0 ° C. and then second drying at 15 ° C.

Efficacy of small dose viral preparations before and after lyophilization virus Efficacy Before Lyophilization
(log PFU / mL) ^* Efficacy after lyophilization
(log PFU / mL) LRSV-404 6.5 6.5 PIV3-cp45 7.7 7.1 LRSV-404 ¹ 6.5 6.3 PIV3-cp45 ¹ 7.7 7.5 (a) ^* = sample frozen at -1 ° C / min
(b) LRSV-404 ¹ ; Efficacy of LRSV-404 ¹ determined from LRSV-404 / PIV-cp45 combination formulation.
(c) PIV-cp45 ¹ ; Efficacy of PIV-cp45 ¹ determined from LRSV-404 / PIV-cp45 combination formulation.

Viral efficacy was tested on the initial viral aggregate, viral material in the vial after the freezing step, and lyophilized samples (immediately after freeze drying). RSV was tested using plaque forming unit (PFU) analysis and Vero cells (ATCC catalog number CCL-18). The assay includes (a) forming cell monolayers in 24-well plates, (b) preparing a 10-fold dilution of the reference and test samples, (c) infecting the cells, (d) 32 ° C. and Incubating the plate at 5% CO ₂ for about 5 days and (e) visualizing the plaques by cell fixation and immunostaining. PIV comprises (a) forming cell monolayers in 24-well plates, (b) preparing 10-fold dilutions of reference and test samples, (c) infecting cells, (d) 32 ° C. and 5 Plaque forming unit (PFU) analysis and LCC-MK2 cells were tested, including incubating the plate for 4 days at% CO ₂ and (e) visualizing the plaques by cell fixation and immunostaining. In these two assays, if the efficacy of the reference sample determined from the assay falls within ± 0.5 log PFU of the recorded value, the results of the efficacy test values are considered to be acceptable.

Efficacy test results for RSV, PIV and RSV / PIV formulations are shown in Table 2 below. From the data it can be seen that there is a minimal loss of efficacy in the frozen formulation at a rate of about -1.0 ° C per minute. The results of this experiment also indicate that RSV and PIV are compatible in the formulation.

Recombinant HA (rHA), HA, Soy Peptone and their at faster freezing rates (-2 ° C./minute; Table 6) and slower freezing rates (−0.3 ° C./minute; Table 3) It was further tested by varying the concentration of the formulation. Virus samples consist of LRSV-404, LRSV-rA38, LRSV-rA42 or PIV3-cp45 liquid virus aggregates prepared for Phase 1 and Phase 2 human clinical trials. Each virus sample was formulated using the formulations exemplified in the second column of Tables 3-6. Virus samples were charged to 2 ml vials (0.5 ml per vial), precooled to a temperature of about 5 ° C., and placed on a lyophilizer shelf. The freeze sample was lyophilized using a cycle comprising first drying at 0 ° C. and then second drying at 15 ° C.

Efficacy tests were performed on the initial vial aggregates, the material of the vial after freezing and the lyophilized sample (immediately after freeze drying). Efficacy test results showed a significant decrease in RSV or PIV efficacy in samples frozen to about -0.3 ° C per minute (Table 3), more of about -1 ° C (Table 4 and Table 5) and -2 ° C (Table 6) per minute. It can be seen that RSV or PIV is very stable in fast frozen formulations.

Efficacy of Small Dose RSV Formulation Frozen at -0.3 ° C Per Minute virus Formulation ¹ Efficacy Before Lyophilization
(log PFU / mL) Efficacy After Freeze Drying
(log PFU / mL) Freezing rate RSV-404 A1 7.1 3.4 -0.3 ° C / min RSV-404 A3 7.2 3.8 -0.3 ° C / min RSV-404 B1 7.1 4.2 -0.3 ° C / min RSV-rA42 A2 6.3 3.9 -0.3 ° C / min RSV-rA42 A4 6.1 3.6 -0.3 ° C / min RSV-rA42 B2 6.3 3.4 -0.3 ° C / min RSV-rA42 C3 6.1 4.9 -0.3 ° C / min RSV-rA42 D2 6.1 4.4 -0.3 ° C / min
Formulation ¹ = Formulations A1-A4, B1, B2, C3 and D2 are described in Example 1.

Efficacy of Small Dose PIV Formulation Frozen at -1 ° C Per Minute virus Formulation ¹ Efficacy Before Lyophilization
(log PFU / mL) Efficacy After Freeze Drying
(log PFU / mL) Freezing rate PIV3-cp45 A1 7.0 6.6 -1 ° C / min PIV3-cp45 A3 7.0 6.7 -1 ° C / min PIV3-cp45 B1 7.0 6.4 -1 ° C / min PIV3-cp45 B3 7.0 6.6 -1 ° C / min PIV3-cp45 C1 7.0 6.5 -1 ° C / min PIV3-cp45 C3 7.0 6.3 -1 ° C / min PIV3-cp45 D1 7.0 6.5 -1 ° C / min
Formulation ¹ = Formulations A1, A3, B1, B3, C1, C3 and D1 are described in Example 1.

Efficacy of Small Dose RSV Formulation Frozen at -1 ° C Per Minute virus Formulation ¹ Efficacy Before Lyophilization
(log PFU / mL) Efficacy After Freeze Drying
(log PFU / mL) Freezing rate LRSV-404 A1 6.6 6.3 -1 ° C / min LRSV-404 C1 6.5 6.0 -1 ° C / min LRSV-rA42 A2 5.9 5.8 -1 ° C / min LRSV-rA42 D2 5.9 6.0 -1 ° C / min LRSV-rA38 A2 5.7 5.4 -1 ° C / min LRSV-rA38 B2 5.4 5.1 -1 ° C / min LRSV-rA38 C4 5.7 5.3 -1 ° C / min LRSV-rA38 D2 5.4 4.8 -1 ° C / min
Formulation ¹ = Formulations A1, A2, B2, C1, C4 and D2 are described in Example 1.

Efficacy of small dose RSV and PIV preparation frozen at -2 ° C per minute virus Formulation ¹ Efficacy Before Lyophilization
(log PFU / mL) Efficacy After Freeze Drying
(log PFU / mL) Freezing rate PIV-cp45 B1 7.3 6.9 -2 ℃ / min LRSV-404 B1 6.3 6.0 -2 ℃ / min LRSV-rA38 B2 5.5 5.5 -2 ℃ / min LRSV-rA38 B4 5.5 5.3 -2 ℃ / min
Formulation ¹ = Formulations B1, B2 and B4 are described in Example 1.

Example  3

Storage stability of small dose formulations containing RSV or PIV

Efficacy was tested at different time points, including 3-month, 6-month, 9-month and 12-month storage at 5 ° C. to evaluate the storage stability of the formulations described in Example 2. The stability data are summarized in Table 7 below, which shows that the loss of efficacy of the viral composition is minimal upon storage at 5 ° C. for up to one year. The formulation column in Table 7 represents the formulation exemplified in Example 1.

Formulation, Freezing Rate and Storage Stability Data for RSV and PIV Compositions Strain
Formulation
virus
aggregate
freezing
speed
^* Lyo Loss
(log PFU)
Efficacy at 5 ° C (log PFU / mL)
1 year
Loss of efficacy
(log PFU)
0 months 3 months 6 months 9 months 12 months LRSV-404 A1 S -1 ° C / min -0.3 6.3 5.9 5.9 5.9 5.5 -0.8 PIV3-cp45 A1 SF -1 ° C / min -0.4 6.6 6.3 5.9 6.0 5.6 -1.0 LRSV-rA42 A2 SF -1 ° C / min -0.1 5.8 5.4 5.2 4.9 4.8 -1.0 PIV3-cp45 B1 SF -1 ° C / min -0.6 6.4 6.2 6.1 5.9 5.7 -0.7 LRSV-rA38 B2 SF -1 ° C / min -0.3 5.1 4.8 4.5 4.2 4.2 -0.9 LRSV-404 C1 S -1 ° C / min -0.0 6.0 6.0 6.2 6.0 5.7 -0.3 PIV3-
cp45 C1 SF -1 ° C / min -0.5 6.5 5.8 6.0 6.2 5.6 -0.9 PIV3-
cp45 C2 SF -1 ° C / min -0.7 6.3 5.7 5.9 5.8 5.5 -0.8 PIV3-
cp45 D1 SF -1 ° C / min -0.5 6.5 5.7 5.4 6.1 5.8 -0.7 LRSV-rA38 D2 SF -1 ° C / min -0.3 4.8 4.5 4.2 4.1 4.3 -0.5
Lyo = Abbreviation for lyophilization
mo = abbreviation for months
S = virus propagates in medium consisting of fetal bovine serum
SF = Proliferation using virus propagation medium "no serum".

Example  4

Effect of freezing rate on high volume RSV and / or PIV formulations during lyophilization

In order to optimize the lyophilization process for mass production of immunogenic compositions comprising RSV, PIV or a combination thereof, large (bulk) RSV or PIV formulations were tested at different freezing rates.

10 mM phosphate buffer at pH 7.0 (2.5 mM HEPES, 0.1 mM magnesium chloride, 0.1 mM calcium chloride, 0.49 mM L (+)-glutamic acid monosodium salt, 50 g / l sucrose and 1 g / l producing a large RSV-404 formulations containing HA), and was freeze-dried in 1-ℓ Lyoguard ^® lyophilization tray. The shelf temperature was reduced from 5 ° C. to −45 ° C. for 45 minutes to freeze material on the lyophilizer shelf. The lyophilized tray was left on the shelf (at −45 ° C.) for another 5 hours to allow the formulation to freeze below the glass transition temperature. The actual time to reach the glass transition temperature (about -35 ° C) is about 2 hours, which corresponds to a freezing rate of -0.3 ° C per minute. Subsequently, a 90 hour lyophilization cycle was performed, including primary drying at 0 ° C. followed by secondary drying at 15 ° C. Initially formulated aggregates and lyophilized material were tested for efficacy by PFU analysis.

In another experiment, a formulation containing LRSV-rA39 was prepared using the same formulation and lyophilized in a 50 ml dose of the same size aluminum tray. The temperature was reduced from 5 ° C. to −40 ° C. for 60 minutes to freeze the material on the lyophilizer shelf. The actual time to reach the glass transition temperature (about -35 ° C) of the material is about 1.5 hours, which corresponds to a freezing rate of -0.4 ° C per minute. Subsequently, a 24-hour lyophilization cycle was conducted, including primary drying at 0 ° C. followed by secondary drying at 15 ° C. Initially formulated aggregates and lyophilized material were tested for efficacy by PFU analysis.

Alternatively, by using large amounts of freeze-dried in a 1-ℓ Lyoguard ^® lyophilization trays were prepared with two different preparations of RSV. LRSV-rA38 and LRSV-404 (proliferated in serum-free medium) were subjected to 12.5 mM HEPES, 0.5 mM magnesium chloride, 0.5 mM calcium chloride, 2.45 mM L (+)-glutamic acid, 50 g / l water Formulated separately in 10 mM phosphate, pH 7.0, containing cross and 10 g / l HA. The tray was immersed in a liquid nitrogen bath for at least 20 minutes to freeze the virus composition. The lyophilization tray is then placed on a precooled (-50 ° C.) lyophilization shelf and (a) initiating primary drying with a vacuum set at 0.10 Torr; (b) temperature rise to a shelf temperature of -23 ° C. (at 0.23 ° C./min); (c) maintaining the temperature at -23 ° C. for 80-100 hours; (d) start secondary drying with vacuum set to 0.02 Torr; (e) temperature rise to a shelf temperature of 15 ° C. (at 0.13 ° C./min); (f) maintaining the temperature at 15 ° C. for 30-40 hours; (g) temperature rise to a shelf temperature of 25 ° C. (at 0.17 ° C./min); And (h) lyophilization in a 120 hour cycle comprising maintaining the temperature at 25 ° C. for 10 hours.

Samples of formulated virus aggregates and lyophilized material were tested for efficacy by PFU analysis. The data in Table 8 below shows that the freezing rate is important for preserving viral efficacy during the lyophilization cycle. Tray freeze on the lyophilized shelf (“slow freeze”) shows a loss of efficacy of the lyophilized material compared to tray cooling with liquid nitrogen (“quick freeze”), where the loss of efficacy is negligible (Table 8, column 3). Significant level (Table 8, column 2).

Effect of Freezing Rate on RSV Efficacy During Mass Lyophilization Efficacy Loss After Mass Lyophilization (log PFU) Strain Freeze in lyophilizer Rapid freezing with liquid nitrogen LRSV-404 -1.2 ND LRSV-rA39 -3.5 ND LRSV-404 ND ^* 0 LRSV-rA38 ND ^* -0.6
ND ^* = not determined

Example 5

Rapid Freezing of Liquid RSV Formulation Filled with Intranasal Spray Apparatus

Liquid formulation of LRSV-rA38 (proliferated in serum free medium) comprises 25 mM HEPES, 1.0 mM magnesium chloride, 1.0 mM calcium chloride, 75 g / L sucrose and 4.9 mM L (+)-glutamic acid Was prepared in 10 mM phosphate buffer solution (pH 7.5). The formulation is filled into a BD Accuspray ^™ intranasal spray device (0.23 ml per device) and each intranasal spray device is inserted into a well of an aluminum intranasal spray holder designed and manufactured by the Applicant (see FIG. 1), intranasally. The spray holder is made of a 96 well aluminum block, where the well diameter is 0.5 mm larger than the diameter of the intranasal spray device. The wells are deep enough so that the viral sample of each intranasal spray device is located below the top of the holder (FIG. 1).

Upon filling the intranasal spray device, a stainless steel plate (dimensions: 0.3 m × 0.2 m × 0.02 m) was placed in a cryocontainer filled with liquid nitrogen and the plate was equilibrated in liquid nitrogen (ie, liquid nitrogen Until boiling stops). After equilibration, the volume of liquid nitrogen in the cold container was adjusted to a volume sufficient to contact the metal plate but not to the intranasal spray holder. An intranasal spray holder comprising a filled intranasal spray device was placed on top of a "freeze" plate in a cold container and "quick frozen" for at least 10 minutes. The intranasal spray device was then removed from the intranasal spray holder to store half of the intranasal spray device in a freezer set at -70 ° C and the other half in a freezer set at -20 ° C.

LRSV-404 liquid formulations (proliferated in serum-free medium) also included 2.5 mM HEPES, 0.1 mM magnesium chloride, 0.1 mM calcium chloride, 75 g / L sucrose and 4.9 mM L (+)-glutamic acid. Was prepared in 10 mM phosphate buffer solution (pH 7.5). The formulation was filled into a BD Accuspray ^™ intranasal spray device (0.23 mL per device), "quickly" frozen and stored as described above.

Alternatively, liquid LRSV-rA38 and liquid LRSV-404 samples were formulated as described above to fill an intranasal spray device and placed in the nasal spray holder on a typical freezer shelf cooled to -70 ° C. to freeze for 24 hours. ("Slow" freeze). The data in FIG. 2 shows the kinetic of “rapid” freeze (■ in FIG. 2) and “slow” freeze (□ in FIG. 2). Half of the intranasal spray device was then stored in a freezer set at -70 ° C and the other half in a freezer set at -20 ° C.

Efficacy was tested at time points 0, 1, 3, 4 and 6 months to assess the storage stability of the samples. Intranasal spray devices (3 devices at each time point) were thawed at room temperature for about 1 hour. The contents of each intranasal spray device were released into tubes and tested for efficacy by PFU analysis.

The data in Tables 9-12 summarize the effect of faster freezing rates on the stability of liquid RSV formulations.

Storage stability (efficacy) of liquid LRSV-rA38 formulations stored at -20 ° C or -70 ° C after freezing at -196 ° C
time
(month) Efficacy (log PFU / mL) Storage temperature -20 ℃ Storage temperature -70 ℃ 0 5.6 5.5 One 5.7 5.5 3 5.6 5.5 4 5.6 5.6 6 5.6 5.6
^** The efficacy of the liquid LRSV-rA38 formulation before freezing at −196 ° C. was 5.6 (log PFU / mL).

Storage stability (efficacy) of liquid LRSV-rA38 formulations stored at -20 ° C or -70 ° C after freezing at -70 ° C
time
(month) Efficacy (log PFU / mL) Storage temperature -20 ℃ Storage temperature -70 ℃ 0 5.1 5.1 One 4.7 5.1 3 5.0 5.0 4 5.2 4.6 6 4.8 5.2
^** The efficacy of the liquid LRSV-rA38 formulation before freezing at -70 ° C was 5.6 (log PFU / mL).

Storage stability (efficacy) of liquid LRSV-404 formulation stored at -20 ° C or -70 ° C after freezing at -196 ° C
time
(month) Efficacy (log PFU / mL) Storage temperature -20 ℃ Storage temperature -70 ℃ 0 5.9 5.9 One 5.8 5.6 3 6.1 5.1 4 6.0 5.9 6 5.9 6.0
^** The efficacy of the liquid LRSV-404 formulation before freezing at −196 ° C. was 6.2 (log PFU / mL).

Storage stability (efficacy) of liquid LRSV-404 formulation stored at -20 ° C or -70 ° C after freezing at -70 ° C
time
(month) Efficacy (log PFU / mL) Storage temperature -20 ℃ Storage temperature -70 ℃ 0 4.1 4.1 One 3.2 3.7 3 5.0 4.5 4 4.9 3.6 6 4.2 3.4
^** The efficacy of the liquid LRSV-404 formulation before freezing at -70 ° C was 6.2 (log PFU / mL).

From the data, it can be seen that the RSV preparation (“rapid” freeze) frozen with liquid nitrogen is stable at both storage temperatures (-20 ° C. and −70 ° C.) (Tables 9 and 11). RSV preparations frozen on the shelf of the freezer at −70 ° C. (“slow” freezing) showed reduced efficacy and severe efficacy fluctuations at different time points (Table 10 and Table 12).

Droplet particle size distribution was measured with a Malvern SprayTec particle size analyzer to evaluate the effect of freezing on spray performance. The analysis was carried out on a spray device filled with the liquid LRSV-rA38 formulation described above. Droplet particle size distribution for the spray device was determined as follows (10 devices per test): (a) intranasal spray device filled with RSV but not frozen, (b) filled with RSV and frozen in liquid nitrogen- Intranasal spray device stored at 70 ° C. for 3 months, (c) filled with RSV, frozen in liquid nitrogen, stored intranasal spray device at -20 ° C. for 3 months, (d) filled with RSV and frozen in freezer, -70 An intranasal spray device stored at 3 ° C. for 3 months and (e) an RSV filled with RSV, frozen in a −70 ° C. freezer and stored at −20 ° C. for 3 months.

Each spray was analyzed because the BD Accuspray ^™ intranasal spray device was designed for intranasal vaccination with two consecutive sprays (separate each nostril). Droplet fraction (%) having a particle size of less than 10 μm was used as a basis (mass increase of fractions less than 10 μm was excluded). The analysis results are summarized in Table 13. Storing the frozen spray device for three months after freezing did not affect spray performance. It was observed that the total mass of the droplets less than 10 μm in diameter did not increase.

Spray performance of the BD Accuspray ^TM device at different conditions Freeze / save
Temperature spray
Count Average Dv (50)
(Μm) Standard Deviation
(Μm) <10 μm fraction
(%) Standard Deviation
(%) Newly charged,
Non-freezing One 123.2 15.7 0.3 0.1 2 168.5 23.2 0.4 0.1 Freeze in liquid nitrogen
Storage at / -70 ℃ One 122.3 15.1 0.2 0.0 2 149.3 20.2 0.2 0.0 Freeze in liquid nitrogen
Storage at / -20 ℃ One 140.7 27.1 0.2 0.1 2 162.1 22.2 0.2 0.1 Freeze at -70 ℃
Storage at / -70 ℃ One 140.8 46.3 0.2 0.1 2 147.3 19.6 0.2 0.0 Freeze at -70 ℃
Storage at / -20 ℃ One 136.8 17.9 0.2 0.1 2 154.6 9.5 0.2 0.0

references

Claims (124)

A process for preparing a storage stable virus composition comprising a virus and a phosphate buffer solution selected from the group consisting of respiratory syncytial virus (RSV), parainfluenza virus (PIV) and combinations thereof, comprising: (a) within a time of up to 60 minutes; Freezing the virus composition below its glass transition temperature at a rate of 0.5 ° C./min to −2.5 ° C./min; And (b) lyophilizing the viral composition, wherein the lyophilized viral composition is stable for at least one year at a storage temperature of 1 ° C to 10 ° C. The method according to claim 1, wherein the glass transition temperature is -40 ° C to -50 ° C. The method according to claim 1, wherein the glass transition temperature is -30 ° C to -40 ° C. The method of claim 3, wherein the glass transition temperature of −35 ° C. is reached within a time of 40 minutes or less. The process according to claim 3, wherein the glass transition temperature of −35 ° C. is reached within 20 minutes or less. The method of claim 1, wherein the viral composition comprises 5.0 mM to 20 mM phosphate buffer solution, the phosphate buffer solution comprises sodium monobasic salt, sodium dibasic salt, potassium monobasic salt or potassium dibasic salt and To a pH of 7.8. The method of claim 6, wherein the viral composition comprises 10 mM phosphate buffer solution. 8. The method of claim 7, wherein said phosphate buffer solution further comprises 0.25 mM to 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). 8. The method of claim 7, wherein said phosphate buffer solution further comprises 0.01 mM to 1 mM magnesium chloride and 0.01 mM to 1 mM calcium chloride. delete The method of claim 9 wherein the phosphate buffer solution is sucrose, L (+)-glutamic acid or L (+)-glutamic acid monosodium salt or a mixture of L (+)-glutamic acid / L (+)-glutamic acid monosodium salt, and A manufacturing method further comprising human albumin (HA). The method of claim 11, wherein the HA is natural or recombinant HA. The method of claim 11, wherein said phosphate buffer solution further comprises soy peptone. 12. The method of claim 11 wherein sucrose is present in an amount of 50 g / l, L (+)-glutamic acid is present in an amount of 0.049 mM to 2.45 mM, and the L (+)-glutamic acid monosodium salt is between 0.049 mM and Wherein the amount is 2.45 mM and HA is present in an amount of 1.0 g / l to 10.0 g / l. The method of claim 14, wherein HA is replaced with 50 g / l Soy Peptone. delete The production method according to claim 1, wherein the storage temperature is 5 ° C. The method of claim 1, wherein the viral composition exhibits a PFU reduction of less than 1.0 log PFU after 1 year of storage at 1 ° C. to 10 ° C. 3. delete The method of claim 1, wherein lyophilizing the viral composition in step (b) comprises 0.2 ml to 1.0 ml of said viral composition in a suitable container means. The method of claim 20, wherein the container means is further defined as a vial, tube or intranasal spray device. The method of claim 1, wherein lyophilizing the viral composition comprises (a) 0.5 ml to 0.6 ml of the viral composition was added to a vial and cooled to a temperature of 5 ° C; (b) placing the vial on a lyophilization shelf to reduce the shelf temperature from 5 ° C. to −50 ° C. at a rate of −1.0 ° C. per minute to −2.0 ° C. per minute; (c) the shelf temperature is maintained at −50 ° C. for 60 minutes; (d) the chamber pressure was reduced to 0.10 Torr and the shelf temperature was maintained at -50 ° C. for 30-60 minutes; (e) the shelf temperature is increased from −50 ° C. to 0 ° C. at a rate of 1.0 ° C. to 2.0 ° C. per minute at 0.10 Torr and the shelf temperature is maintained at 0 ° C. for 540 minutes to 720 minutes; (f) the shelf temperature is increased from 0 ° C. to 15 ° C. at a rate of 0.5 ° C. per minute at 0.10 Torr and the shelf temperature is maintained at 15 ° C. for 600 to 720 minutes; (g) Filling the vial with nitrogen gas to further seal it. The method of claim 1, wherein lyophilizing the viral composition comprises (a) 0.5 ml to 0.6 ml of the viral composition was added to a vial and cooled to a temperature of 5 ° C; (b) freezing the lyophilized shelf to a temperature of −70 ° C .; (c) placing the vial on a lyophilized shelf to maintain the temperature at −70 ° C. for 60 minutes; (d) reducing the chamber pressure to 0.10 Torr and increasing the shelf temperature from -70 ° C to -50 ° C at a rate of 1.0 ° C per minute; (e) the shelf temperature is increased from −50 ° C. to 0 ° C. at a rate of 1.0 ° C. to 2.0 ° C. per minute at 0.10 Torr and the shelf temperature is maintained at 0 ° C. for 540 minutes to 720 minutes; (f) the shelf temperature is increased from 0 ° C. to 15 ° C. at a rate of 0.5 ° C. per minute at 0.10 Torr and the shelf temperature is maintained at 15 ° C. for 600 to 720 minutes; (g) Filling the vial with nitrogen gas to further seal it. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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