FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
AND
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See Section 10; Rule 13)
METHOD FOR MANUFACTURING VIRAL VACCINES AND COMPOSITIONS
THEREOF
SERUM INSTITUTE OF INDIA PVT. LTD.
an Indian Company
of 212/2, Off Soli Poonawalla Road,
Hadapsar, Pune-411028,
Maharashtra, India
THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED.
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FIELD
The present disclosure relates to method of producing clarified virus pool and manufacturing of
viral vaccines, more particularly, manufacturing a lyophilized/freeze-dried live attenuated virus
vaccine composition/formulation comprising of Measles, Mumps, Rubella
antigens/immunogens or combinations thereof.
BACKGROUND
All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Measles is an acute viral illness caused by Morbillivirus from family paramyxovirus. Measles is characterized by a prodrome of fever (as high as 105°F) and malaise, cough, coryza, and conjunctivitis, followed by a maculopapular rash. Rash spreads from head to trunk to lower extremities. Measles is usually a mild or moderately severe illness with possibility of further complications as pneumonia, encephalitis, and death. Approximately, one case of encephalitis and two to three deaths occur for every 1,000 reported measles cases.
In rare cases, Measles infection is also followed by subacute sclerosing panencephalitis (SSPE), a fatal disease of the central nervous system that generally develops 7 to 10 years after infection. Among persons who contracted measles during the resurgence in the United States (U.S.) in 1989 to 1991, the risk of SSPE was estimated to be 7 to 11 cases/100,000 cases of measles. The risk of developing SSPE be higher when measles occurs prior to the second year of life. The average incubation period for measles is 11 to 12 days, and the average interval between exposure and rash onset is 14 days, with a range of 7 to 21 days. Persons with measles are usually considered infectious from four days before until four days after onset of rash with the rash onset being considered as day zero.
Before the introduction of measles vaccine in 1963 and widespread vaccination, major epidemics occurred approximately every 2 to 3 years and measles caused an estimated 2.6 million deaths each year. More than 140 000 people died from measles in 2018 – mostly children under the age of 5 years, despite the availability of a safe and effective vaccine.
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Accelerated immunization activities have had a major impact on reducing measles deaths. During 2000 to 2018, measles vaccination prevented an estimated 23.2 million deaths. Global measles deaths have decreased by 73% from an estimated 536 000 in 2000 to 142 000 in 2018. According to the World Health Organization (WHO), the largest outbreak of measles was seen in India in 2022 with reported measles cases of 12,773 making India’s goal to eliminate measles by 2023 to be impractical. There was a recent outbreak of measles in Maharashtra with 3075 cases and 13 deaths. As per WHO, more than 58 000 people in 41 of the 53 Member States in the Region – straddling Europe and central Asia – were infected with measles, in 2023, resulting in thousands of hospitalizations and 10 measles-related deaths.
Measles is still common in many developing countries – particularly in parts of Africa and Asia. The overwhelming majority (more than 95%) of measles deaths occur in countries with low per capita incomes and weak health infrastructures. Measles outbreaks can be particularly deadly in countries experiencing or recovering from a natural disaster or conflict. Damage to health infrastructure and health services interrupts routine immunization and overcrowding in residential camps greatly increases the risk of infection.
Mumps is an acute viral illness caused by a paramyxovirus that typically presents as swelling of the parotid (parotitis) or another salivary gland[s]. Parotitis occurs as unilateral or bilateral and lasts from 3 to 7 days (average 5 days); most cases resolve within 10 days. In some cases, nonspecific prodromal symptoms precede parotitis by several days, including low-grade fever, lasting 3 to 4 days, myalgia, anorexia, malaise, and headache. The incubation period ranges from 12 to 25 days, but parotitis typically develops 16 to 18 days after exposure to mumps virus.
Mumps can occur in a person who is fully vaccinated, but vaccinated persons are at much lower risk for mumps and mumps complications. Mumps reinfection in patients who previously had natural infection or recurrent mumps (parotid swelling resolves and then weeks to months later occurs on the same or other side) can also occur. Mumps infection may present only with nonspecific or primarily respiratory symptoms or be asymptomatic. Among unvaccinated people, approximately 20% of infections are asymptomatic; frequency of asymptomatic infection among vaccinated people is unknown.
Worldwide, mumps is not as well controlled as measles and rubella. From 1999 to 2019, on average, about 500,000 mumps cases were reported to the World Health Organization annually;
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however, global mumps incidence is challenging to estimate as mumps is not a notifiable disease in many countries. As of 2019, mumps vaccine is routinely used in 122 of 194 (63%) countries. Since the mid-2000s, mumps outbreaks have also been reported among populations with high 2-dose MMR coverage in other countries, including United Kingdom, Ireland, New Zealand, Canada, Netherlands, Spain and Norway. Despite these outbreaks, mumps incidence is still much higher in countries that do not have routine mumps vaccination.
Rubella is an acute contagious viral infection that occurs most often in children and young adults. Rubella is the leading vaccine-preventable cause of birth defects. While rubella virus infection usually causes a mild fever and rash in children and adults, infection during pregnancy, especially during the first trimester, can result in miscarriage, Foetal death, stillbirth, or infants with congenital malformations, known as congenital rubella syndrome (CRS). The rubella virus is transmitted by airborne droplets when infected people sneeze or cough. Humans are the only known host. There is no specific treatment for rubella but the disease is preventable by vaccination.
Rubella is a viral illness caused by a Togavirus of the genus Rubivirus and is characterized by a mild, maculopapular rash. The rubella rash occurs in 80% of rubella-infected persons and is sometimes misdiagnosed as measles or scarlet fever. Children usually develop few or no constitutional symptoms, but adults may experience a 1 to 5-day prodrome of low-grade fever, headache, malaise, mild coryza, and conjunctivitis. Postauricular, occipital and posterior cervical lymphadenopathy is characteristic and precedes the rash by 5 to 10 days. Arthralgia or arthritis may occur in up to 70% of adult women with rubella. Rare complications include thrombocytopenic purpura and encephalitis. Rubella is transmitted through direct or droplet contact from nasopharyngeal secretions and has an average incubation period of 17 days (range: 12 to 23 days). Persons with rubella are most infectious when rash is erupting, but they can shed virus from 7 days before to 7 days after rash onset.
Measles and rubella vaccination are an integral part of immunization programmes worldwide, contributing to progress towards achieving global immunization goals and, more broadly, the Global Health Security Agenda and the United Nations Sustainable Development Goals (SDGs).
The Region of the Americas, which was verified to have eliminated measles in 2016, lost its measles elimination status in 2018. Globally, reported measles cases more than doubled from
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2017 to 2018, from 170 000 to 350 000. This upward trend continued into 2019, with several countries experiencing large measles outbreaks. In 2019, the Democratic Republic of the Congo, Ukraine and Brazil reported 333 017, 57 282 and 18 203 confirmed cases of measles, respectively, while Chad reported more than 26 600 suspected cases. Vaccination coverage remains low or very low in several countries. In 2019, seven countries had coverage of the first dose of measles-containing vaccine (MCV1) below 50% and 23 had coverage below 70%, indicating that 30–50% of children in these countries had not received any doses of measles vaccine through routine service delivery mechanisms.
To achieve the goal of providing full immunization coverage of more than 90% and to reach the goal of universal immunization program, India has launched an Intensified Mission Indradhanush (IMI) 4.0 in phases in 2022. Although the prevention of measles and rubella is a priority, according to the recommendations of FOGSI [Federation of Obstetric and Gynecological Societies of India (FOGSI)], the combination of MMR vaccine is preferred over rubella vaccine for the purpose of routine preconception vaccination. Despite the availability of MMR vaccines, recent outbreaks of measles and a higher prevalence of mumps are alarming and warrant a broader MMR vaccination coverage in the country.
The worldwide market demand for MMR vaccines is in the order of approximately 110 million doses per year. Efficient vaccine production requires the growth of large-scale quantities of virus produced in high yields from a host system. The process and cultivation conditions under which a virus strain is grown is of great significance with respect to achieving an acceptable high yield of the strain. Thus, in order to maximize the yield of the desired virus, both the system and the cultivation conditions must be adapted specifically to provide an environment that is advantageous for the production of the desired virus. Therefore, a continuing need exists for safe and effective methods to produce viruses and antigen. Moreover, there is a need for an approach to viral propagation, employing materials that are already available and requiring a minimal number of time-consuming manipulations, wherein the selection of a combination of host cells, culture medium, growth conditions and production system is essential to achieve an efficient production process.
However, live attenuated vaccines are particularly fragile and their vaccinating action is quickly destroyed when they are exposed to temperatures above + 4 ° C and sometimes even less. Such vaccine instability is unacceptable, especially when these vaccines are used, on a large scale in tropical countries where they may accidentally be exposed to the high ambient temperatures
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prevailing in these countries. It is therefore necessary to have a stable and heat-resistant vaccine capable of withstanding deterioration when it is stored for long periods of time and is accidentally exposed to high temperatures.
Vaccine stability depends on various factors such as such as storage temperature, storage time, the vaccine composition, as well as the surrounding gas and the vial in which the vaccine is kept. Vaccines tend to lose their effectiveness over time when stored under ambient or warm temperatures. Hence there is a need to develop methods of vaccine composition which elicits a protective immune response with improvements in stability and shelf life of the vaccines.
There are various disadvantages linked to the use of serum and of animal-derived components, mainly their cost, the batch to batch variability in their composition, their association with a higher contamination risk by adventitious agents, and the subsequent difficulties encountered in downstream processing (e.g. purification to get rid of the serum- proteins or of the introduced animal-derived proteins).
Before manufacturing-scale mammalian cell cultivation process starts in a bioreactor, a seed culture inoculum is typically prepared. This involves culturing production cells in a series of single or multi-plate flasks in incubators and/or smaller bioreactors of increasing volume until enough cells are available for inoculation into the production bioreactor. The process involves transferring a cell population from one culture vessel to a larger one. Generally, a 20% to 50% dilution of the cell population is used for each transfer or subculture. In the incubator, the flasks with culture medium are stationary or clamped to a rotating platform to swirl the culture and facilitate gas transfer between the culture medium and the atmosphere in the incubators. Typically, the incubator for a mammalian cell culture process is set at 37℃ with 5% carbon dioxide (CO2) and a humidity level higher than about 80%. Similar temperatures and CO2 levels are used for seed cultures grown in bioreactors. When the seed culture reaches a sufficient volume and cell density, it is inoculated into the production flask/ bioreactor.
After seed culture is inoculated into the bioreactor medium, parameters such as pH, temperature, and level of dissolved oxygen are controlled to the prescribed levels during the cell cultivation process. pH is typically controlled by adding basic or acidic solutions when necessary during the process. Commonly used base solutions include sodium bicarbonate, sodium carbonate and sodium hydroxide solutions. Dissolution of carbon dioxide (CO2) is commonly used to achieve a more acidic pH. Although other acids are available for controlling
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pH, the dissolved CO2 and sodium bicarbonate combination forms a most stable and favourable buffer system for the cell culture. The preferred temperature of the culture medium or solution for mammalian cell cultivation processes is about 37℃. The desired level of dissolved oxygen in the culture medium or solution is typically achieved through air sparging using sparger installed on the bottom of the bioreactor or through headspace, along with agitation of the culture medium or solution using impellers which breakup the large air/oxygen bubbles to enhance the transfer of oxygen to the cell medium from the sparged air bubbles or using any other gaseous exchanger systems. Purging the bioreactor headspace with a cover gas provides a limited degree of surface gas exchange. Disadvantageously, air-sparging and agitation of the culture medium may result in foaming and shear damage to the mammalian cells which adversely impacts cell viability. Accumulations of foam on the surface of the culture medium also serve to further limit surface gas exchange and to reduce the available working volume of the bioreactor.
Mammalian cells are known to be sensitive to the amount of dissolved carbon dioxide in the cell culture media. Mammalian cell cultures exposed to excess carbon dioxide levels during the exponential growth phase may demonstrate reduced production of monoclonal antibodies or other desired biological products. Before inoculation, the pH of the slightly alkaline culture media has to be lowered with carbon dioxide adjusted to an optimum value. This often leads to elevated levels of dissolved carbon dioxide at the beginning of the lag phase of many mammalian cell culture processes. Dissolved carbon dioxide in mammalian cell culture bioreactors originates from chemical and biological sources. The chemical source of carbon dioxide is equilibrium chemical reactions occurring within the cell culture medium that includes a selected amount of a buffer solution containing sodium bicarbonate and/or sodium carbonate. Additionally, carbon dioxide may be directly sparged into the slightly alkaline culture medium to reduce the pH of the broth to a prescribed level, usually around 7.0, resulting in more dissolved carbon dioxide. The biological source of carbon dioxide is a product of the respiration of the mammalian cells within the bioreactor. This biological source of carbon dioxide increases with cell density and generally reaches its maximum value at about the same time that cell density within the bioreactor is maximized. However, as more carbon dioxide is produced, the pH of the cell culture medium trends toward acidic such that additional bicarbonate is needed to keep the pH of the cell culture medium or solution within the desired range. To offset the effects of increased dissolved carbon dioxide which depresses the pH, one may add sodium bicarbonate so as to maintain the pH of the solution within the prescribed
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range. Both of these means to offset the effects of increased carbon dioxide have other negative consequences on the mammalian cell culture process.
The addition of sodium bicarbonate, needed to adjust the pH of the solution to offset the carbon dioxide, also increases osmolality. (Osmolality represents the number of dissolved particles per kilogram of solution and is commonly reported as mOsm/kg by freeze-point depression). The addition of sodium bicarbonate will also increase the equilibrium saturation level of dissolved carbon dioxide allowed in the solution, making carbon dioxide more difficult to be removed during the aeration process. It is known in the art that increased levels of either dissolved carbon dioxide or increased osmolality have adverse or negative impacts on cell density or yield.
Carbon dioxide dissociates into bicarbonate ions at a pH of 7 in water. Only a fraction of the carbon dioxide remains as free CO2 in an un- dissociated state. Removing the dissolved carbon dioxide from a cell culture thus becomes difficult as most mammalian cell cultures take place at pH in the range of 6.5 to 7.5. The dissociated bicarbonate ions are not easily removed and generally must be recombined into free carbon dioxide before they can be stripped out of the solution.
The conventional method of removing or stripping dissolved carbon dioxide from a mammalian cell culture solution is by sparging the cell culture solution with air or a gas mixture of air/oxygen/nitrogen in agitated tanks. However, gas sparging in agitated tanks results in adverse effects to the cell culture process. In particular, the gas-bubble breakage at the tip of the rotating agitator is a source of high shear rate that damages mammalian cell membranes, often sufficiently to cause cell death. Even when damage is sub-lethal, cell productivity is compromised in the period that the damaged membrane is repaired.
Also, sparging air or nitrogen into the bioreactor creates gas bubbles rising to the surface of the solution within the bioreactor where the gas is released into the headspace. Gas bubble breakage at the top surface of the cell culture solution is often more damaging to the mammalian cells than the damage caused by the agitator. Restraining the agitator speed and limiting the gas sparging rate are currently viewed as the best means to avoid such damage and increase cell viability. However, these measures reduce the amount of carbon dioxide that can be removed and the excess that cannot be removed also inhibits cell growth and viability. These disadvantages are particularly challenging to overcome in large, commercial- scale bioreactors
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where the shear rate goes up substantially with the diameter of the impellers. Also, the greater hydrostatic head of large-scale bioreactors tends to increase the solubility of carbon dioxide, meaning that more carbon dioxide needs to be removed to maintain dissolved CO2 levels within an optimal range.
As per the above explained parameters, there is need of manufacturing process which can overcome the issues related to the process parameters/steps involved in the manufacturing of measles vaccine.
Accordingly, need exist for large scale, cost effective and safe method of producing viral vaccines that comprises minimum animal origin components, provide high virus yield, ensures virus structure integrity/stability preservation across manufacturing and storage.
Applicant has surprisingly found improved large scale affordable /safe manufacturing processes (encompassing cultivation, purification & formulation stages) that utilizes minimum animal origin components, provides high virus yield, ensures virus structure integrity/ stability preservation across manufacturing and storage wherein 1) Recombinant Trypsin (at least 95% pure, having molecular weight 23 to 25 kilodalton; Specific activity 2500 USP units/mg; low endotoxin content) at optimal pH (7.2 to 7.6) is used; 2) > 50% virus yield obtained (increased by 30-40 %) due to optimal pH (7.2 to 7.8), optimal concentration of sodium bicarbonate in medium (1.0 to 2.5 g/L), Low multiplicity of infection (MOI) i.e. virus to cell ratio (for measles and rubella bulk vaccine manufacturing – 1:5 to 1: 20 and for mumps bulk vaccine manufacturing – 1:20 to 1: 60), multiple harvesting; 3) use of Gamma irradiated FBS (Dose range of 20 to 50KGy); 4) optimum virus: Stabilizer-I: Stabilizer-II ratio (80:20:10); 5) use of MLTCF-10 vented with Corning vented caps (instead of milex filter) resulting in slow dissociation of sodium bicarbonate.
OBJECTS
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows:
An object of the present disclosure is to ameliorate one or more problems of the prior art or to at least provide a useful alternative.
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An object of the present disclosure is to provide clarified virus pool in the manufacturing of live attenuated lyophilized/freeze dried virus vaccine which includes one or more than one virus.
Another object of the present disclosure is to provide improved method for manufacturing live attenuated lyophilized viral vaccine composition.
Another object of the present disclosure is to provide improved method for manufacturing live attenuated lyophilized viral vaccine composition comprising atleast one virus selected from a group consisting of a Measles, Mumps and Rubella.
Another object of the present disclosure is to provide easy and simple method for manufacturing live attenuated viral vaccine bulk which may contribute to the increase in the final yield of bulk vaccine and doses.
Another object of the present disclosure is to provide easy and simple method for manufacturing live attenuated lyophilized viral vaccine composition which may contribute to the increase in the final yield of bulk vaccine and doses.
Still another object of the present disclosure is to provide a method for manufacturing live attenuated lyophilized viral vaccine composition using Gamma irradiated FBS.
Still another object of the present disclosure is to provide a method for manufacturing live attenuated lyophilized viral vaccine composition using recombinant trypsin at optimal pH.
Yet another object of the present disclosure is to provide a method for manufacturing live attenuated lyophilized viral vaccine composition with high virus yield using optimal pH, optimal concentration of sodium bicarbonate in medium, low MOI i.e. virus to cell ratio and multiple harvesting.
Still another object of the present disclosure is to provide a method for manufacturing live attenuated lyophilized viral vaccine composition using MLTCF-10 vented with Corning vented caps (instead of milex filter).
Yet another object of the present disclosure is to provide a live attenuated lyophilized viral vaccine composition containing optimum virus: stabilizer ratio.
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Another object of the present disclosure is to provide a live attenuated lyophilized/freeze-dried viral vaccine composition/formulation wherein, post-reconstitution the composition preserves the desired characteristics of the virus, including stability and immunogenicity.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.
SUMMARY
Applicant provides a method of producing a clarified virus pool, the method comprising: providing a cell line in a cell media and supplement; treating cells of cell line with enzyme; infecting the cell line with a virus to form an infected cell line; washing of the infected cell line with the virus media; harvesting the infected cell line in the media to obtain a harvest; optionally re-harvesting the infected cell line; adding a stabilizer to the harvest; and clarifying the harvest to obtain a clarified virus pool (CVP).
In an aspect, the present invention is directed to a method of producing a clarified virus pool, the method comprising:
a. providing a cell line in a cell media, a buffer and a supplement;
b. treating cells of the cell line with at least one enzyme;
c. infecting the cell line with a virus to form an infected cell line;
d. washing of the infected cell line with a virus media and the buffer;
e. harvesting the infected cell line in the media to obtain a harvest; optionally re-harvesting
the infected cell line one or more times;
f. adding at least one stabilizer to the harvest; and
g. clarifying the harvest to obtain a clarified virus pool (CVP).
optionally, wherein the cell line or the infected cell line in exponential growth phase is
- grown with additional ventilation or aeration, or
- provided with re-addition of the buffer or both.
In another aspect, the present invention is directed to a clarified virus pool (CVP) obtained by the method as disclosed.
In another aspect, the present invention is directed to a method of producing a measles clarified virus pool.
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In another aspect, the present invention is directed to a measles clarified virus pool (CVP) obtained by the method as disclosed.
In another aspect, the present invention is directed to a method of producing a mumps clarified virus pool.
In another aspect, the present invention is directed to a mumps clarified virus pool (CVP) obtained by the method as disclosed.
In another aspect, the present invention is directed to a method of producing a rubella clarified virus pool.
In another aspect, the present invention is directed to a rubella clarified virus pool (CVP) obtained by the method as disclosed.
In another aspect, the present invention is directed to method of obtaining a lyophilized/ freeze-dried Measles, Mumps, Rubella (MMR) immunogenic composition comprising one or more CVP selected from a measles CVP, a mumps CVP, a rubella CVP, or a combination thereof, blending of CVP, followed by lyophilizing the blended CVP.
In another aspect, the present invention is directed to lyophilized/ freeze-dried MMR immunogenic composition.
In another aspect, the present invention is directed to kit comprising the lyophilized/freeze-dried MMR immunogenic composition.
Applicant provides a method of producing a clarified virus pool of viruses such as measles, mumps, rubella. The enzyme treatment done for cells of cell line is recombinant trypsin. Washing of infected cell line is done with virus medium without FBS. The buffer added in cell media and virus medium is selected NaHCO3 (sodium bicarbonate). Sodium bicarbonate in virus medium maintains the physiological pH which gives the significant outcomes. Applicant has found the improved large scale affordable/ safe manufacturing processes (encompassing cultivation, purification & formulation stages) that utilize minimum animal origin components, provide high virus yield, ensure virus structure integrity/stability preservation across manufacturing & storage wherein 1) Recombinant Trypsin (at least 95% pure, having molecular weight 23 to 25 kilodalton; Specific activity 2500 USP units/mg; low endotoxin content) at optimal pH (7.2 to 7.6) is used; 2) > 50% virus yield obtained (increased by 30 to 40
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%) due to optimal pH (7.2 to 7.8), optimal concentration of sodium bicarbonate in medium (1.0 to 2.5 g/L) Low MOI i.e. virus to cell ratio (for measles and rubella bulk vaccine manufacturing – 1:5 to 1: 20 and for mumps bulk vaccine manufacturing – 1:20 to 1: 60), multiple harvesting; 3) use of Gamma irradiated FBS (Dose range of 20 to 50KGy); 4) Optimum virus: Stabilizer-I: Stabilizer-II ratio (80:20:10); 5) use of MLTCF-10 vented with Corning vented caps (instead of milex filter) resulting in slow dissociation of sodium bicarbonate.
DETAILED DESCRIPTION OF THE DRAWING
The present invention will now be described with the help of the accompanying drawing, in which:
Figure 1 illustrates yield of Measles virus.
DESCRIPTION
Although the present disclosure may be susceptible to different embodiments, certain embodiments are shown in the drawing and following detailed discussion, with the understanding that the present disclosure can be considered an exemplification of the principles of the disclosure and is not intended to limit the scope of disclosure to that which is illustrated and disclosed in this description.
Embodiments are provided so as to thoroughly and fully convey the scope of the present disclosure to the person skilled in the art. Numerous details are set forth, relating to specific components, and processes, to provide a complete understanding of embodiments of the present disclosure. It will be apparent to the person skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the present disclosure. In some embodiments, well-known composition, well-known processes, and well-known techniques are not described in detail.
The terminology used, in the present disclosure, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present disclosure. As used in the present disclosure, the forms “a”, “an”, and “the” may be intended to include the plural forms as well, unless the context clearly suggests otherwise.
The terms “comprises”, “comprising”, “including”, and “having” are open ended transitional phrases and therefore specify the presence of stated features, integers, steps, operations,
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elements, modules, units and/or components, but do not forbid the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The particular order of steps disclosed in the process of the present disclosure is not to be construed as necessarily requiring their performance as described or illustrated. It is also to be understood that additional or alternative steps may be employed.
The terms first, second, third, etc., should not be construed to limit the scope of the present disclosure as the aforementioned terms may be only used to distinguish one element, component, region, layer or section from another component, region, layer or section. Terms such as first, second, third etc., when used herein do not imply a specific sequence or order unless clearly suggested by the present disclosure.
It is understood that each feature or embodiment, or combination, described herein is a non-limiting, illustrative example of any of the aspects of the invention and, as such, is meant to be combinable with any other feature or embodiment, or combination, described herein. For example, where features are described with language such as “one embodiment”, “some embodiments”, “certain embodiments”, “further embodiment”, “specific exemplary embodiments”, and/or “another embodiment”, each of these types of embodiments is a non-limiting example of a feature that is intended to be combined with any other feature, or combination of features, described herein without having to list every possible combination. Such features or combinations of features apply to any of the aspects of the invention.
Definitions:
In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth throughout the specification.
The term “PDL” is “population doubling level” refers to the total number of times the cells in a given population have doubled during in vitro culture.
The term “working cell bank” refers to quantity of cells of uniform compositions derived from the master cell bank at a finite passage level, dispensed in aliquots into individual containers appropriately stored in liquid nitrogen containers at -196°C, one or more of which would be used for production purposes.
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The term “cell media” may be interchangeably used with the term “cell growth media”.
The term “virus media” may be interchangeably used with the term “virus growth media” and “washing media”
The terms “CF” refers to “cell factory” and “CS” refers to “cell stack”.
The terms MLTCF refers to “Multilayered Tissue culture flask”.
The term “working seed virus” refers to the virus used for infecting the cell line.
The term “freeze-drying/ freeze dried/lyophilize/ lyophilization” involves lyophilization and refers to the process by which a suspension/solution is frozen, after which the water is removed by sublimation at low pressure.
The term “sublimation” refers to a change in the physical properties of a composition, wherein the composition changes directly from a solid state to a gaseous state without becoming a liquid.
The terms “Tresivac” refers to “Measles, Mumps, Rubella Vaccine, Live Attenuated (Freeze-Dried) (MMR vaccine) of Serum Institute of India, Ltd”.
The term “Blind vaccine” refers to diluent used for dilution of virus bulk or clarified virus pool/ pools during blending or used for mixing with virus bulk or clarified virus pool/ pools during blending.
The term “bulk vaccine” refers to the solution containing the clarified virus pool of virus or the blended solution of containing the clarified virus pool of more than one virus.
The terms “bulk vaccine” or may be interchangeably used with the term “virus bulk vaccine”
The term "dose" herein is typically one administration of the vaccine of the invention, which is typically one injection. A typical human dose is 0.5mL. Of course, various doses may be administered in a vaccine administration schedule.
The present disclosure envisages a method for manufacturing viral vaccines. In a preferred embodiment, the present disclosure provides a method for manufacturing of live attenuated lyophilized/freeze dried virus vaccine which includes one or more than one virus. The lyophilized/freeze dried virus vaccine include the bulk of one or more virus vaccine.
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The present disclosure provides a method of producing a clarified virus pool in the manufacturing of live attenuated lyophilized/freeze dried virus vaccine which includes one or more than one virus.
In a preferred embodiment, the present disclosure provides a method of producing a clarified virus pool in the manufacturing of live attenuated lyophilized/freeze dried virus vaccine which includes one or more than one virus increases virus titre by addition of media in the manufacturing process.
In a preferred embodiment, the present disclosure provides a method of producing a clarified virus pool in the manufacturing of live attenuated lyophilized/freeze dried virus vaccine which includes one or more than one virus increases virus titre by addition of media post infection with virus in the manufacturing process.
In a preferred embodiment, the present disclosure provides a method of producing a clarified virus pool in the manufacturing of live attenuated lyophilized/freeze dried virus vaccine which includes one or more than one virus increases virus yield by addition of media post infection with virus in the manufacturing process.
In a preferred embodiment, the present disclosure provides a method of producing a clarified virus pool in the manufacturing of live attenuated lyophilized/freeze dried virus vaccine which includes one or more than one virus increases viral load by addition of media post infection with virus in the manufacturing process.
In a preferred embodiment, the present disclosure provides a method of producing a clarified virus pool in the manufacturing of live attenuated lyophilized/freeze dried virus vaccine which includes one or more than one virus increases final yield of bulk vaccine and doses by addition of media post infection with virus in the manufacturing process.
In a preferred embodiment, the present disclosure provides a method of producing a clarified virus pool in the manufacturing of live attenuated lyophilized/freeze dried virus vaccine which includes one or more than one virus increases virus titre, virus yield, final yield of bulk vaccine and doses by addition of media post infection with virus in the manufacturing process.
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GENERAL CVP PRODUCTION METHOD
In an aspect of the present disclosure, the method in accordance with the present disclosure comprise:
a. providing a cell line in a cell media, a buffer and a supplement;
b. treating cells of the cell line with at least one enzyme;
c. infecting the cell line with a virus to form an infected cell line;
d. washing of the infected cell line with a virus media and the buffer;
e. harvesting the infected cell line in the media to obtain a harvest; optionally re-harvesting
the infected cell line;
f. adding atleast one stabilizer to the harvest; and
g. clarifying the harvest to obtain a clarified virus pool (CVP).
optionally, wherein the cell line or the infected cell line in exponential growth phase is
- grown with additional ventilation or aeration, or
- provided with additional buffer or
- both.
In an embodiment, the method of producing the CVP is for preparation of bulk virus vaccine/ bulk virus immunogenic composition.
In another embodiment, the method for producing CVP includes:
a) Providing a cell line depending on the virus;
b) Revival & passaging of cells in Tissue culture flask (TCF);
c) Preparation of cell factories/ cell stacks;
d) Treating cells of the cell line with at least one enzyme, the enzyme including trypsin, i.e. Trypsinization of cell factories/ cell stacks;
e) Infecting the cell line by addition of virus i.e. infection;
f) Washing of the infected cell line using he virus media and the buffer;
g) Harvesting the infected cell line in the media to obtain the harvest; re-harvesting the infected cell lines to obtain multiple harvests;
h) Adding atleast one stabilizer to the harvest;
i) Clarifying of the harvest to obtain the clarified virus pool (CVP) j) Storing the CVP below -20°C as a drug substance i.e. bulk virus vaccine optionally, wherein the cell line or the infected cell line in exponential growth phase is
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- grown with additional ventilation or aeration, or
- provided with additional buffer or
- both.
STEP a):
In an embodiment, the method of producing the clarified virus pool includes providing the cell line in the cell media, the buffer and the supplement.
In an embodiment, the cell line is provided in a container. The container includes covered flasks, bottles, roller bottles, serrated roller bottles, cuboidal containers, round bottom containers, cell factories.
In an embodiment, the container is covered. The covering for container includes caps.
CELL LINE
In another embodiment, the cell line used as a substrate for the virus growth includes animal cell line, insect cell line, human cell line, primary cell line (Chick embryo fibroblast (CEF)), diploid cell line (including Human Lung Fibroblast (MRC-5)), continuous cell line.
In an embodiment, the cell line used as a substrate for the virus growth is selected from the different cell lines such as Rhesus monkey kidney (RhMK) cells; Primary rabbit kidney cells; Human foreskin fibroblasts; Chick embryo fibroblast (CEF); Human epidermoid carcinoma cells (HEp-2); Human lung carcinoma cells (A549); Human Cervix Epithelial (HeLa); African Green Monkey Kidney Epithelial (Vero); Human Lung Fibroblast (MRC-5); Human Lung Fibroblast (MRC-9); Mouse Embryo Fibroblast (NIH3T3); Mouse Connective Tissue Fibroblast (L929); Chinese Hamster Ovary Fibroblast (CHO); Syrian Hamster Kidney Fibroblast (BHK-21); Human embryo Kidney Epithelial (HEK-293); Human Liver Epithelial (HepG2); Bovine Aorta Endothelial (BAE-1); Human Neuroblastoma Neuronal (SH-SY5Y); Mouse Myeloma Lymphoblast (NS0); Human Hystiocytic Lymphoma Lymphoblast (U937); Human Leukemia Lymphoblast (HL60); Mouse B-cell Lymphoma Lymphoblast (WEHI231); Mouse Lymphoma Lymphoblast (YAC1); Human Myeloma Lymphoblast (U266B1); Human T-cell Leukemia Lymphoblast (Jurkat); Human Monocyte Leukemia Lymphoblast (THP-1); Human embryonic lung cells (W1-38); Madin Darby canine kidney cells (MDCK); Human embryonic retinal cells (PER.C6); Human embryonic retinoblasts (HER.911); Murine non-secreting myeloma (Sp2.0); Epithelial cells of African green monkey kidney origin (BSC-1
18

cells); Rhesus Monkey Kidney Epithelial Cells (LLC-MK2 cells); Cercopithecus aethiops monkey kidney cells (CV-1 cells); African green monkey kidney fibroblast-like cells (COS-cells); Crandell- Rees Feline Kidney Cells (CRFK cells); Rapidly Accelerated Fibrosarcoma cells (RAF cells); Normal Rabbit Kidney Epithelial Cells (RK-13 cells); Transformed C3H Mouse Kidney-1 (TCMK-1 cells); Pig Kidney Epithelial Cells (LLC-PK1 cells); Porcine kidney cells (PK15 cells); Rabbit kidney cell line (LLC-RK1 cells), Nonsecreting myeloma cell lines (NS-1 cells), New human male diploid cell strain (TIG-1, TIG-7); nonhuman primate diploid cell line (FRhL-2); Human foetal lung (IMR-90, IMR-91) cells; human diploid lung fibroblasts (including WI-38) and others.
In an embodiment, the cell line used as a substrate for the growth of measles virus, mumps virus, rubella virus is selected from Human Lung Fibroblast (MRC-5) cell line and Chick embryo fibroblast (CEF) cell line.
In another embodiment, MRC-5 cells at lower passage were obtained from NIBSC, UK. The master and working cells banks are prepared and stored at –196°C in liquid nitrogen. This stock of cells is used for production purposes. The cell stock has been characterized and tested according to the Ph. Eur and WHO.
In another embodiment, Chick Embryo Fibroblast (CEF) cells is prepared by using 9 to 11 days old embryo of specific pathogen free (SPF) chicken eggs. These SPF eggs are received from Lohmann Germany and Hy-Vac, USA etc.
In an embodiment, the cell line is provided with the cell media, the supplement and the buffer.
CELL MEDIA
In an embodiment, for the method of producing the CVP, the cell line is provided with a cell media (herein after interchangeably referred to as cell growth media) for growth and propagation. The cell media has nutrients including carbon sources, carbohydrates, vitamins, amino acids, minerals, growth factors, hormones, sugars, glucagon, cyclodextrin, inorganic salts, buffers or combination thereof.
The carbon source includes carbohydrates, glucose, glutamine, sucrose, dextrose, galactose, fructose or combinations thereof.
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In another embodiment, the cell growth medium is selected from basal medium, enriched medium, selective and indicator medium, transport media and storage media.
In another embodiment, the cell growth medium is selected from different media such as basal culture media, cell culture media well suited for the growth of a broad spectrum of mammalian cells, Minimum Essential Medium supplemented with L- Glutamine, multipurpose media, minimum essential medium modified to contain Balanced Salt Solution (BSS) and amino acids.
In another embodiment, the Balanced salt solution includes Earle's balanced salt solution (EBSS), Gey's balanced salt solution (GBSS), Hanks' balanced salt solution (HBSS), (Dulbecco's) Phosphate buffered saline (PBS), Puck's balanced salt solution, Ringer's balanced salt solution (RBSS), Alsever's solution, Simm's balanced salt solution (SBSS), TRIS-buffered saline (TBS), Tyrode's balanced salt solution (TBSS). In a preferred embodiment, the Balanced Salt Solution is Earle’s or Hanks’s BSS.
In an embodiment, the cell growth medium is selected from Medium 199 (M–199), Dulbecco’s Modified Eagle’s Medium (DMEM), Minimum Essential Medium (MEM); Eagle’s Minimum Essential Medium (E–MEM), Hank’s MEM (H–MEM), Iscove’s Modified Dulbecco’s Medium (IMDM), Ham’s nutrient mixtures (F–10 and F–12), Leibovitz (L–15), Roswell Park Memorial Institute (RPMI)–1640; Neurobasal medium; Schneider's Drosophila medium; McCoy's 5A Medium; Dynamis medium; Essential 8 (E8) media; StemFlex culture media; Airway Epithelial Cell basal medium; alpha-modified minimum essential medium (α-MEM); StemMacs iPS-Brew media; TeSR-E8, mTeSR1, mTeSR Plus medium; GMEM(Glasgow Minimum Essential Medium); Opti-MEM I; SmGM-2; fibroblast growth media / FGM; StemPro-34 serum free growth medium; mTESR1 medium; ECGM-2 media; Williams’ Medium E; Medium M254; CnT07 media; TNM-FH media; mammary epithelial cell growth basal medium (MEBM); complete skeletal muscle media and NeuroCult NS-A basal medium.
In another embodiment, the cell growth medium as synthetic cell culture medium is Minimum Essential Medium. The medium is sterilized by using sterile grade hydrophilic filters.
In a preferred embodiment, the filtration of the medium is done by 0.1µ sterile grade hydrophilic filters.
In another embodiment, cell growth medium is supported or added with the buffer and the supplements.
20

In another embodiment, the cell growth medium is Minimum Essential Medium (MEM) which is prepared using powdered MEM supplemented with L-Glutamine, Foetal bovine serum (FBS) and pH buffer by mixing all the ingredients in pre-sterilized water for injection. The medium is sterilized by 0.1µ filtration.
BUFFER
In an embodiment, for the method of producing the CVP, the buffer is used.
The buffer includes NaHCO3, NaOH, NaCl, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) i.e HEPES, (piperazine-N,N′-bis(2-ethanesulfonic acid)) i.e. PIPES, or (2-(N-morpholino) ethanesulfonic acid), i.e. MES.
In an embodiment, the buffer includes NaHCO3 (sodium bicarbonate).
In a preferred embodiment, the buffer, i.e. NaHCO3 (sodium bicarbonate) is in used in a batch in concentration range from 0.5 to 4.0 g/L, or 0.8 to 3.5 g/L or 1.0 to 3.0 g/L or 1.0 to 2.5 g/L or 1.0 to 2.3 g/L, or 1.0 to 2.1 g/L, or 1.0 to 2.0 g/L.
In a more preferred embodiment, the buffer, i.e. NaHCO3 (sodium bicarbonate) is in used in a batch in concentration range from 1.0 to 1.9 g/L, or 1.2 to 1.9 g/L.
In another embodiment, the infected cell line is washed with the virus media and the buffer.
In another embodiment, the cell line or the infected cell line in exponential growth phase is grown with re-addition of the buffer.
In another embodiment, the cell line or the infected cell line in exponential growth phase is grown with additional ventilation or aeration and re-addition of the buffer.
In another embodiment the re-addition of the buffer is performed by boosting with additional buffer, i.e. NaHCO3 or providing additional amount of the buffer with the cell media or the virus media or both.
In an embodiment, the boosted buffer concentration for the cell media or the virus media or both during the re-addition of the buffer is in the range of 5% to 40% or 5% to 35% or 5% to 30%.
In an embodiment, the boosted buffer concentration for the cell media or the virus media or both during the re-addition of the buffer is in the range of 10% to 30% or 15% to 30%.
21

In an embodiment, the boosted buffer concentration for the cell media or the virus media or both during the re-addition of the buffer is in the range of 0.1 g/L to 1.6 g/L or 0.1 g/L to 1.2 g/L or 0.1 g/L to 1.0 g/L or 0.1 g/L to 0.8 g/L or 0.1 g/L to 0.6 g/L.
In an embodiment, the total buffer concentration including the boosted buffer concentration for the cell media or the virus media or both is in the range of 0.6 g/L to 6.0 g/L, or 0.8 g/L to 6.0 g/L, or 1.0 g/L to 6.0 g/L, or 1.2 g/L to 6.0 g/L in the batch.
In another embodiment, the total buffer concentration for the cell media or the virus media or both including the boosted buffer concentration is in the range of 1.2 g/L to 5.9 g/L, or 1.2 g/L to 5.8 g/L, or 1.2 g/L to 5.7 g/L, or 1.2 g/L to 5.6 g/L in the batch.
In preferred embodiment, the total buffer concentration for the cell media or the virus media or both including the boosted buffer concentration is in the range of 1.5 g/L to 2.0 g/L in the batch.
In an embodiment, the method of preparing the CVP includes providing the cells of the cell line with the cell media, the buffer and at least one supplement.
The pH is critical process parameters for growth of cells. pH is controlled by proper venting to allow gaseous exchange and increasing the buffering capacity of cell media by adding additional quantity of the buffer in the cell media which cause no fluctuation in the pH, increase the cell count and increase the yield.
Thus, pH is one of the critical process parameters in viral vaccine manufacturing process. Exposure of the virus to extremes of pH inactivates the virus. It has been reported that virus is stable at pH in the range from 6 to 8 and a progressive inactivation of virus occurs on either side. Physical stability of virus is highly compromised in acidic environment. Even slight changes in pH affects the native confirmation of the virus.
Regulation of pH is particularly important immediately following cell seeding when a new culture is establishing and is usually achieved by one of two buffering systems; (i) a “natural” buffering system where gaseous CO2 balances with the CO3/ HCO3 content of the culture medium and (ii) chemical buffering using a zwitterion called HEPES. Cultures using natural bicarbonate /CO2 buffering systems need to be maintained in an atmosphere of 5-10% CO2 in air usually supplied in a CO2 incubator. Bicarbonate/CO2 is low cost, non-toxic and also
22

provides other chemical benefits to the cells. HEPES has superior buffering capacity in the pH range 7.2-7.4 but is relatively expensive and can be toxic to some cell types at higher concentrations. HEPES buffered cultures do not require a controlled gaseous atmosphere.
In an embodiment, growth of cells is be controlled by adding additional quantity of NaHCO3 in the cell media which cause no fluctuation in the pH, increase the cell count and increase the yield. pH as one of the critical process parameters for growth of cells is be controlled by proper venting to allow gaseous exchange and increasing the buffering capacity of cell media by adding additional quantity of NaHCO3 in the cell media which cause no fluctuation in the pH, increase the cell count and increase the yield.
SUPPLEMENTS
In an embodiment, the at least one supplement includes additional amino acids, cholesterol, proteins, lactoferrin, linoleic acid, glucagon, cyclodextrin, yeast extracts, serums, antioxidants, vitamins, buffers, antibiotics, nutrients, trace elements, adherence, and extension factors.
In another embodiment, the at least one supplement includes serum, antibiotics, additional amino acid.
In another embodiment, the at least one supplement includes serum, antibiotics.
In another embodiment, the at least one supplement includes antibiotics, additional amino acid.
In another embodiment, the at least one supplement includes serum, additional amino acid.
In an embodiment, serum as supplement is bovine serum or calf serum. Serum as supplement is foetal bovine serum or foetal calf serum. Serum as supplement is foetal bovine serum.
Foetal bovine serum, (FBS) is the most widely used basal media supplement for in vitro cell culture. It contains very low level of antibodies and high concentration of growth factors such as hormones, attachment factors and transport proteins. FBS is used to keep cells alive for longer period of time. FBS contains a large number of components, like growth factors, proteins, vitamins, trace elements, hormones which are essential for the growth and maintenance of cells. FBS effectively promotes and sustains the growth of cells at low densities used for biological manufacturing. Serum also adds buffering capacity to the medium and binds or neutralizes toxic components. FBS is obtained from foetuses harvested in abattoirs from
23

healthy animals. Serum lots from the manufacturer contain pools of serum collected from many different animals.
Being an animal origin material, FBS has an inherent risk of being contaminated with transmissible adventitious agents. Bovine serum might be contaminated by many different bovine viruses. It is evident that each serum batch has to be tested for those viruses which are ubiquitous and of known risk. The possibility of the introduction and replication of adventitious agents during cell culture has long been recognized as a potential risk which leads to virus contaminated final product. There have been a number of instances where laboratory studies provided evidence for the presence of adventitious agents in marketed vaccines. Such risks of adventitious agents are typically mitigated by testing bovine serum for absence of adventitious agents. Most regulatory bodies allow the use of animal derived materials only when their use can be justified due to absence of viable alternative. For that, each serum lot must be tested for sterility and adventitious agents. Virus testing is typically performed in accordance with various regulatory guidelines. However, the testing methods have their own limitations and sometimes the contaminating adventitious agent may escape detection.
FBS undergo post-manufacturing treatment methods as follows;
1. Filtration: Triple 0.1 μm filtration has become the standard method of aseptic processing for various types of serum. While this methodology will remove bacteria and most mollicutes, it cannot render serum free of all viruses. Nanofiltration using viral retentive filters (20 nm nominal pore size) can be effective for removal of even small viruses such as parvoviruses. However, it suffers from scalability and flux decay with serum materials. Therefore, nanofiltration is not suitable for large-scale processing of serum product.
2. UV Treatment: Ultraviolet irradiation (typically 254 nm) has been shown to be effective for pathogen reduction. The method is not only effective for larger microbes such as bacteria and mollicutes but is also very effective for most viruses. It is commonly employed for high-volume water disinfection. UV irradiation has also demonstrated efficacy in small-scale experiments aimed at clearing adventitious agents from cell culture media. However, it has not yet been optimized to effectively handle the larger volumes of media needed for commercial-scale bioproduction, and further technology development is needed.
24

WE CLAIM:
1. A method of producing a clarified virus pool, the method comprising:
a. providing a cell line in a cell media, a buffer and a supplement;
b. treating cells of the cell line with at least one enzyme;
c. infecting the cell line with a virus to form an infected cell line;
d. washing of the infected cell line with a virus media and the buffer;
e. harvesting the infected cell line in the media to obtain a harvest; optionally re-
harvesting the infected cell line one or more times;
f. adding at least one stabilizer to the harvest; and
g. clarifying the harvest to obtain a clarified virus pool (CVP).
optionally, the cell line or the infected cell line in exponential growth phase is
- grown with additional ventilation or aeration, or
- provided with re-addition of the buffer or
- both.

2. The method as claimed in claim 1, wherein the virus is selected from single-stranded, positive-sense, negative-sense, enveloped, non-enveloped RNA viruses belonging to the family Picornaviridae, Caliciviridae, Togaviridae, Matonaviridae, Flaviviridae, Coronaviridae, Retroviridae, Filoviridae, Bunyaviridae, Rhabdoviridae, Orthomyxoviridae, Arenaviridae, Paramyxoviridae.
3. The method as claimed in claim 1 or 2, wherein the virus is single-stranded, negative-sense, enveloped, RNA viruses belonging to the family Paramyxoviridae and single-stranded, positive-sense, enveloped, RNA viruses belonging to the family Matonaviridae.
4. The method as claimed in any one of the claims 1 to 3, wherein the virus is Measles morbillivirus (measles virus), Mumps orthorubulavirus (mumps virus) and Rubivirus rubellae (rubella virus).
5. The method as claimed in any one of the claims 1 to 4, wherein the cell line is selected from animal cell line, mammalian, avian, insect cell line, human cell line, primary cell line, diploid cell line, continuous cell line, Rhesus monkey kidney (RhMK) cells; Primary rabbit kidney cells; Human foreskin fibroblasts; Chick embryo fibroblast (CEF); Human epidermoid carcinoma cells (HEp-2); Human lung carcinoma cells (A549); Human Cervix Epithelial (HeLa); African Green Monkey Kidney Epithelial (Vero); Human Lung Fibroblast (MRC-5); Human Lung Fibroblast (MRC-9); Mouse Embryo Fibroblast (NIH3T3); Mouse Connective Tissue Fibroblast (L929); Chinese Hamster Ovary
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Fibroblast (CHO); Syrian Hamster Kidney Fibroblast (BHK-21); Human embryo Kidney Epithelial (HEK-293); Human Liver Epithelial (HepG2); Bovine Aorta Endothelial (BAE-1); Human Neuroblastoma Neuronal (SH-SY5Y); Mouse Myeloma Lymphoblast (NS0); Human Hystiocytic Lymphoma Lymphoblast (U937); Human Leukemia Lymphoblast (HL60); Mouse B-cell Lymphoma Lymphoblast (WEHI231); Mouse Lymphoma Lymphoblast (YAC1); Human Myeloma Lymphoblast (U266B1); Human T-cell Leukemia Lymphoblast (Jurkat); Human Monocyte Leukemia Lymphoblast (THP-1); Human embryonic lung cells (W1-38); Madin Darby canine kidney cells (MDCK); Human embryonic retinal cells (PER.C6); Human embryonic retinoblasts (HER.911); Murine non-secreting myeloma (Sp2.0); Epithelial cells of African green monkey kidney origin (BSC-1 cells); Rhesus Monkey Kidney Epithelial Cells (LLC-MK2 cells); Cercopithecus aethiops monkey kidney cells (CV-1 cells); African green monkey kidney fibroblast-like cells (COS-cells); Crandell- Rees Feline Kidney Cells (CRFK cells); Rapidly Accelerated Fibrosarcoma cells (RAF cells); Normal Rabbit Kidney Epithelial Cells (RK-13 cells); Transformed C3H Mouse Kidney-1 (TCMK-1 cells); Pig Kidney Epithelial Cells (LLC-PK1 cells); Porcine kidney cells (PK15 cells); Rabbit kidney cell line (LLC-RK1 cells), Nonsecreting myeloma cell lines (NS-1 cells), New human male diploid cell strain (TIG-1, TIG-7); nonhuman primate diploid cell line (FRhL-2); Human foetal lung (IMR-90, IMR-91) cells; human diploid lung fibroblasts and others.
6. The method as claimed in any one of the claims 1 to 5, wherein the cell line is selected from Human Lung Fibroblast (MRC-5) cell line and Chick embryo fibroblast (CEF) cell line.
7. The method as claimed in any one of the claims 1 to 6, wherein the enzyme is selected from trypsin, recombinant trypsin, dipase, collagenase, hyaluronidase, elastase, cysteine protease, deoxyribonuclease I and chymotrypsin.
8. The method as claimed in any one of the claims 1 to 7, wherein the cell media includes basal media, enriched media, selective and indicator media, transport media, storage media, carbon sources, or combination thereof.
9. The method as claimed in any one of the claims 1 to 8, wherein the supplement includes amino acids, cholesterol, proteins, lactoferrin, linoleic acid, yeast extracts, serums, antioxidants, vitamins, antibiotics, nutrients, trace elements, adherence agents, extension factors, or combinations thereof.
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10. The method as claimed in any one of the claims 1 to 9, wherein the buffer is selected from the NaHCO3, NaOH, NaCl, HEPES, PIPES, MES, phosphates, carbonates, Hank’s, Earl’s or combinations thereof.
11. The method as claimed in any one of the claims 1 to 10, wherein the virus media for washing the infected cell line is selected from basal media, enriched media, selective and indicator media, transport media, storage media, carbon sources, or combination thereof.
12. The method as claimed in any one of the claims 1 to 11, wherein the washing of the infected cell lines by the virus media is performed post infection with virus having MOI in the range of 1:5 to 1: 60 and is followed by incubation at 30 to 40°C.
13. The method as claimed in any one of the claims 1 to 12, wherein the at least one stabilizer includes at least one carbohydrate, at least one protein, at least one amino acid, or a combination thereof.
14. The method as claimed in any one of the claims 1 to 13, wherein the clarification of the harvest is carried out using filters, preferably the filters are of material of construction selected from modified PVDF, cellulose acetate, polyethersulfone, polypropylene.
15. The method as claimed in any one of the claims 1 to 14, wherein the clarification of the harvest is carried out using at least one filter having pore size in the range of 0.1 to 10.0 µ.
16. The clarified virus pool (CVP) obtained by the method as claimed in any one of the claims 1 to 15.
17. The method as claimed in any one of the claims 1 to 16, for producing a measles clarified virus pool, the method comprising:
a. providing the cell line in a cell media, the buffer and the supplement;
b. treating cells of the cell line with the enzyme;
c. infecting the cell line with Measles morbillivirus to form the measles infected cell
line;
d. washing of the measles infected cell line with the virus media and the buffer;
e. harvesting the measles infected cell line in the media to obtain the harvest; optionally
re-harvesting the measles infected cell line;
f. adding the stabilizer to the harvest; and
g. clarifying the harvest to obtain the measles clarified virus pool (CVP).
optionally, wherein the cell line or the measles infected cell line in exponential growth phase is
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- grown with additional ventilation or aeration, or
- provided with re-addition of the buffer or
- both.

18. The method as claimed in claim 17, wherein the cell line is Human Lung Fibroblast (MRC-5) cell line.
19. The method as claimed in any one of the claims 17 or 18, wherein the enzyme is recombinant trypsin.
20. The method as claimed in any one of the claims 17 to 19, wherein the cell media is Minimum Essential Medium.
21. The method as claimed in any one of the claims 17 to 20, wherein the supplement includes glutamine and foetal bovine serum.
22. The method as claimed in claim 21, wherein foetal bovine serum is in the range 5% to 15%.
23. The method as claimed in claim any one of the claims 17 to 22, wherein the virus media is Minimum Essential Medium.
24. The method as claimed in any one of the claims 17 to 23, wherein the buffer comprises of sodium bicarbonate added to virus media and the buffer is in the range 0.5 to 4.0 g/L to maintain optimal pH of 6 to 8.
25. The method as claimed in any one of the claims 17 to 24, wherein the measles virus includes Edmonston strain, including the Schwartz, the Edmonston-Zagreb, the Moraten strains; CAM-70; TD 97; Leningrad-16; AIK-C strain and Shanghai 191 (Ji-191) strains.
26. A measles clarified virus pool (CVP) obtained by the method as claimed in any one of claims 17 to 25.
27. The method as claimed in any one of the claims 1 to 16 for producing a mumps clarified virus pool, the method comprising:
a. providing the cell line in the cell media, the buffer and the supplement;
b. treating cells of the cell line with the enzyme;
c. infecting the cell line with the Mumps orthorubula virus to form a mumps infected
cell line;
d. washing of the mumps infected cell line with the virus media and the buffer;
e. harvesting the mumps infected cell line in the media to obtain the harvest; optionally
re-harvesting the mumps infected cell line;
f. adding the stabilizer to the harvest; and
g. clarifying the harvest to obtain the mumps clarified virus pool (CVP).
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optionally, wherein the cell line or the mumps infected cell line in exponential growth phase is
- grown with additional ventilation or aeration, or
- provided with re-addition of the buffer or
- both.

28. The method as claimed in claim 27, wherein the cell line is Chick embryo fibroblast (CEF) cell line.
29. The method as claimed in claim 27 or 28, wherein the enzyme is recombinant trypsin.
30. The method as claimed in any one of the claims 27 to 29, wherein the cell media is Minimum Essential Medium.
31. The method as claimed in any one of the claims 27 to 30, wherein the supplement includes glutamine, foetal bovine serum, and neomycin sulphate.
32. The method as claimed in claim 31, wherein foetal bovine serum is in the range 5% to 15%.
33. The method as claimed in any one of the claims 27 to 32, wherein the virus media is Minimum Essential Medium.
34. The method as claimed in any one of the claims 27 to 33, wherein the buffer is sodium bicarbonate added to virus media and the buffer is in the range 0.5 to 4.0 g/L to maintain optimal pH of 6 to 8.
35. The method as claimed in any one of the claims 27 to 34, wherein the virus includes Jeryl-Lynn, RIT 4385, Leningrad-3, Leningrad-Zagreb (L-Zagreb), Urabe Am9, Hoshino strain, Torii strain and S79 Rubini strains.
36. A mumps clarified virus pool (CVP) obtained by the method as claimed in any one of the claims 27 to 35.
37. The method as claimed in any one of the claims 1 to 16 for producing a rubella clarified virus pool, the method comprising:
a. providing the cell line in the cell media and the supplement;
b. treating cells of the cell line with the enzyme;
c. infecting the cell line with the Rubivirus rubellae to form a rubella infected cell line;
d. washing of the rubella infected cell line with the virus media and the buffer;
e. harvesting the rubella infected cell line in the media to obtain the harvest; optionally
re-harvesting the rubella infected cell line;
f. adding the stabilizer to the harvest; and
g. clarifying the harvest to obtain the rubella clarified virus pool (CVP).
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optionally, wherein the cell line or the rubella infected cell line in exponential growth phase is
- grown with additional ventilation or aeration, or
- provided with re-addition of the buffer or
- both.

38. The method as claimed in claim 37, wherein the cell line is Human Lung Fibroblast (MRC-5) cell line.
39. The method as claimed in claim 37 or 38, wherein the enzyme is recombinant trypsin.
40. The method as claimed in any one of claims 37 to 39, wherein the cell media is Minimum Essential Medium.
41. The method as claimed in any one of the claims 37 to 40, wherein the supplement includes glutamine, foetal bovine serum, neomycin sulphate or combination thereof.
42. The method as claimed in claim 41, wherein foetal bovine serum is in the range 5% to 15%.
43. The method as claimed in any one of the claims 37 to 42, wherein the virus media is Minimum Essential Medium.
44. The method as claimed in any one of the claims 37 to 43, wherein the buffer is sodium bicarbonate added to virus media and the buffer is in the range 0.5 to 4 g/L to maintain optimal pH of 6 to 8.
45. The method as claimed in any one of the claims 37 to 44, wherein the virus includes Wister RA 27/3 strain, BRD-2 strain, Matsuba, DCRB19, Takahashi, Matsuura and TO-336 strains.
46. A rubella clarified virus pool (CVP) obtained by the method as claimed in any one of claims 37 to 45.
47. A method of obtaining a lyophilized/ freeze-dried MMR immunogenic composition comprising at least one CVP selected from a measles CVP, a mumps CVP, a rubella CVP or a combination thereof, the method comprising:
providing a measles CVP of claim 26 or obtained by any one of claims 17 to 25, a mumps CVP of claim 36 or obtained by any one of claim 27 to 35, a rubella CVP of claim 46 or obtained by any one of claims 37 to 45, or a combination thereof, blending of the CVPs, followed by lyophilizing the blended CVPs.
48. The method as claimed in claim 47, wherein the method includes:
a) thawing at least one CVP of measles, mumps, rubella virus or combination thereof at 30 to 35°C to obtain the thawed CVP;
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b) blending the thawed CVP and blind vaccine to obtain a blended solution;
c) clarifying the blended solution through a 0.45µ filter to obtain a homogenous bulk;
d) aseptically filling the homogenous bulk into sterilized vials, followed by transferring the vials to a lyophilizer/ freeze dryer.
e) lyophilizing/ freeze drying the vials containing the homogenous bulk.
49. The method as claimed in claim 48, wherein the lyophilizing/ freeze-drying step
comprises:
a) pre-freezing shelf, loading the trays containing vials of the MMR immunogenic composition on the shelf;
b) freezing;
c) primary drying/ sublimation and
d) secondary drying
50. A lyophilized/ freeze-dried MMR immunogenic composition obtained by the method as
claimed in claim 49, the composition comprising;
a) atleast one virus;
b) stabilizer comprising atleast one carbohydrate, atleast one amino acid and atleast one hydrolyzed protein.

51. The lyophilized/ freeze-dried MMR immunogenic composition as claimed in claim 50, comprising of live attenuated measles virus present at a dose of not less than 1000 CCID50 per dose, live attenuated mumps virus present at a dose of not less than 5000 CCID50 per dose and live attenuated rubella virus present at a dose of not less than 1000 CCID50 per dose.
52. The lyophilized/ freeze-dried MMR immunogenic composition as claimed in claim 50, comprising of atleast one carbohydrate selected from a group consisting of natural carbohydrate, synthetic carbohydrate, monosaccharides, disaccharides, trisaccharides, oligosaccharides, reducing sugar, non-reducing sugar, sugar alcohols, polyol, polyhydroxyl compounds, chemically modified carbohydrates and glass transition facilitating agents which include sucrose, mannitol, trehalose, mannose, raffinose, lactitol, lactobionic acid, glucose, maltulose, iso- maltulose, maltose, lactose sorbitol, dextrose, fructose, glycerol, sorbitol, and fucose and a combination thereof.
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53. The lyophilized/ freeze-dried MMR immunogenic composition as claimed in claim 50, comprising of atleast one amino acid selected from a group consisting of tricine, leucine, iso-leucine, L-histidine, glycine, glutamine, L-arginine, L-arginine hydrochloride, lysine, L-alanine, Tryptophan, Phenylalanine, Tyrosine, Valine, Cysteine, Glycine, Histidine, Methionine, Proline, Serine, Threonine and a combination thereof.
54. The lyophilized/ freeze-dried MMR immunogenic composition as claimed in claim 50, comprising of atleast one hydrolyzed protein obtained by chemical, enzymatic or thermal hydrolysis of protein from either plant or animal sources.
55. The lyophilized/ freeze-dried MMR immunogenic composition as claimed in claim 50, comprising of at least one hydrolyzed protein selected from a group consisting of gelatin, lactalbumin hydrolysate, monosodium glutamate, collagen hydrolysate, keratin hydrolysate, peptides, Casein hydrolysate and whey protein hydrolysate.
56. The lyophilized/ freeze-dried MMR immunogenic composition as claimed in claim 50, wherein the composition comprises;

a) at least one carbohydrate at concentration range of 1-20% (w/v)
b) at least one amino acid at concentration range of 0.01-10% (w/v);
c) at least one hydrolyzed protein at concentration range of 0.1-10% (w/v)

57. The lyophilized/ freeze-dried MMR immunogenic composition as claimed in claim 56, wherein at least one of the carbohydrate is sorbitol present at a concentration of 1 to 20% (w/v), 1 to 10% (w/v), preferably 3-6% (w/v).
58. The lyophilized/ freeze-dried MMR immunogenic composition as claimed in claim 56, wherein at least one of the amino acid selected from a group consisting of tricine present at a concentration of 0.1% to 2% (w/v), L-histidine present at a concentration of 0.1% to 2% (w/v), L-alanine present at a concentration of 0.01% to 1% (w/v) and L-arginine hydrochloride present at a concentration of 0.1% to 5% (w/v).
59. The lyophilized/ freeze-dried MMR immunogenic composition as claimed in claim 56, wherein atleast one of the hydrolysed protein selected from a group consisting of gelatin present at a concentration of 0.1% to 5% (w/v) and lactalbumin hydrolysate present at a concentration of 0.1% to 2% (w/v).
60. The lyophilized/ freeze-dried MMR immunogenic composition as claimed in claim 50, comprising of an adjuvant selected from a group consisting of aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, and potassium aluminum sulfate or a mixture thereof.
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61. The lyophilized/ freeze-dried MMR immunogenic composition as claimed in claim 50, comprising of an immunostimulatory component selected from a group consisting of an oil and water emulsion, MF-59, a liposome, a lipopolysaccharide, a saponin, lipid A, lipid A derivatives, Monophosphoryl lipid A, 3–deacylated monophosphoryl lipid A, AS01, AS03, an oligonucleotide, an oligonucleotide comprising at least one unmethylated CpG and/or a liposome, Freund’s adjuvant, Freund’s complete adjuvant, Freund’s incomplete adjuvant, CRL-8300 adjuvant, muramyl dipeptide, TLR-4 agonists, flagellin, flagellins derived from gram negative bacteria, TLR-5 agonists, fragments of flagellins capable of binding to TLR-5 receptors, QS-21, ISCOMS, Chitosan, saponin combination with sterols and lipids.
62. The lyophilized/ freeze-dried MMR immunogenic composition as claimed in claim 50, comprising of a pharmaceutically acceptable additive selected from a group consisting of transporter, excipient, binder, carrier, isotonic agent, emulsifier and humectant.
63. The lyophilized/ freeze-dried MMR immunogenic composition as claimed in claim 50, wherein the excipient is selected from a group consisting of salt including NaCl, KCl, KH2PO4, Na2HPO4.2H2O, CaC12, and MgCl2; non-ionic surfactant including polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer - 0.001%-0.05%; polymers including dextran, carboxymethylcellulose, hyaluronic acid ad cyclodextrin.
64. The lyophilized/ freeze-dried MMR immunogenic composition as claimed in claim 50, wherein the lyophilized/freeze-dried viral vaccine composition is reconstituted with an aqueous solution selected from a group consisting of saline, buffer and WFI (water for injection).
65. The lyophilized/ freeze-dried MMR immunogenic composition as claimed in claim 64, wherein the buffer is selected from a group consisting of sodium chloride, acetate, carbonate, citrate, lactate, gluconate, tartrate, phosphate buffer saline, borate, histidine buffer, succinate buffer, HEPES, TRIS and Citrate-phosphate.
66. The lyophilized/ freeze-dried MMR immunogenic composition as claimed in claim 64, wherein the final pH of the reconstituted composition is in the range of pH 6.5 to 7.5.
67. The lyophilized/freeze-dried MMR immunogenic composition as claimed in any one of the claims 50-66, comprising:
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a) live attenuated measles virus present at a dose of not less than 1000 CCID50 per dose, live attenuated mumps virus present at a dose of not less than 5000 CCID50 per dose and live attenuated rubella virus present at a dose of not less than 1000 CCID50 per dose;
b) stabilizer comprising
carbohydrate consisting of sorbitol present at a concentration of 1 to 10% (w/v); amino acid consisting of tricine present at a concentration of 0.1% to 2% (w/v), L-histidine present at a concentration of 0.1% to 2% (w/v), L-alanine present at a concentration of 0.01% to 1% (w/v) and L-arginine hydrochloride present at a concentration of 0.1% to 5% (w/v); and
hydrolyzed protein consisting of gelatin present at a concentration of 0.1% to 5% (w/v) and lactalbumin hydrolysate present at a concentration of 0.1% to 2% (w/v).
68. The lyophilized/freeze-dried MMR immunogenic composition as claimed in claim 67,
comprising:
a) live attenuated measles virus present at a dose of not less than 1000 CCID50 per dose, live attenuated mumps virus present at a dose of not less than 5000 CCID50 per dose and live attenuated rubella virus present at a dose of not less than 1000 CCID50 per dose;
b) stabilizer comprising
carbohydrate consisting of sorbitol present at a concentration of 5% (w/v);
amino acid consisting of tricine present at a concentration of 0.3% (w/v), L-histidine
present at a concentration of 0.21% (w/v), L-alanine present at a concentration of
0.1% (w/v) and L-arginine hydrochloride present at a concentration of 1.6% (w/v);
and
hydrolyzed protein consisting of gelatin present at a concentration of 2.5% (w/v) and
lactalbumin hydrolysate present at a concentration of 0.35% (w/v).
69. The lyophilized/freeze-dried MMR immunogenic composition as claimed in claim 68, wherein the lyophilized virus vaccine composition is in the form of a single dose composition or a multi-dose composition.
70. The lyophilized/freeze-dried MMR immunogenic composition as claimed in claim 69, wherein the multi-dose composition additionally comprises preservative.
71. A kit comprising the lyophilized/freeze-dried MMR immunogenic composition as claimed in claim 67 or 68 comprises;
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a) a first container containing a lyophilized (freeze-dried) viral vaccine composition said composition comprising: live attenuated measles virus present at a dose of not less than 1000 CCID50 per dose, live attenuated mumps virus present at a dose of not less than 5000 CCID50 per dose and live attenuated rubella virus present at a dose of not less than 1000 CCID50 per dose; carbohydrate consisting of sorbitol present at a concentration of 1 to 10% (w/v); amino acid consisting of tricine present at a concentration of 0.1% to 2% (w/v), L-histidine present at a concentration of 0.1% to 2% (w/v), L-alanine present at a concentration of 0.01% to 1% (w/v) and L-arginine hydrochloride present at a concentration of 0.1% to 5% (w/v); and hydrolyzed protein consisting of gelatin present at a concentration of 0.1% to 5% (w/v) and lactalbumin hydrolysate present at a concentration of 0.1% to 2% (w/v); and
b) a second container containing an aqueous solution selected from saline or water for injection (WFI) for the reconstitution of the lyophilized (freeze-dried) vaccine composition.
72. A kit comprising the lyophilized/freeze-dried MMR immunogenic composition as claimed in claim 67 or 68 comprises;
a) a first container containing a lyophilized (freeze-dried) viral vaccine composition said composition comprising: live attenuated measles virus present at a dose of not less than 1000 CCID50 per dose, live attenuated mumps virus present at a dose of not less than 5000 CCID50 per dose and live attenuated rubella virus present at a dose of not less than 1000 CCID50 per dose; carbohydrate consisting of sorbitol present at a concentration 5% (w/v); amino acid consisting of tricine present at a concentration 0.3% (w/v), L-histidine present at a concentration 0.21% (w/v), L-alanine present at a concentration 0.1% (w/v) and L-arginine hydrochloride present at a concentration 1.6% (w/v); and hydrolyzed protein consisting of gelatin present at a concentration of 2.5% (w/v) and lactalbumin hydrolysate present at a concentration 0.35% (w/v); and
b) a second container containing an aqueous solution selected from saline or water for injection (WFI) for the reconstitution of the lyophilized (freeze-dried) vaccine composition.
Dated this 27th day of March 2023 ^AM^H^
Archana Shanker (IN/PA-149)
Of Anand and Anand Advocates
Agents for the Applicants