Method of manufacturing viral vaccines and their compositions

Description

1 / 156 “METHOD OF MANUFACTURING VIRAL VACCINES AND THEIR COMPOSITIONS” FIELD

[001] The present invention relates to a method of producing a pool of clarified viruses and to the manufacture of viral vaccines, more particularly, to the manufacture of a freeze-dried / freeze-dried live attenuated virus vaccine composition / formulation comprising measles, mumps and rubella antigens / immunogens or combinations thereof. BACKGROUND

[002] All publications contained herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated for incorporation by reference. The following description includes information useful for understanding the present invention. It is not an admission that any information provided herein is prior art or relevant to the invention now claimed, or that any specific or implicitly referenced publication is prior art.

[003] Measles is an acute viral disease caused by Morbillivirus of the paramyxovirus family. Measles is characterized by a prodrome of fever (up to 40 °C) and malaise, cough, runny nose, and conjunctivitis, followed by a maculopapular rash. The rash spreads from the head to the trunk and lower extremities. Measles is generally a mild to moderately severe disease, with the possibility of further complications such as pneumonia, encephalitis, and death. Approximately one case of encephalitis and two to three deaths occur for every 1,000 reported cases of measles.

[004] In rare cases, measles infection is also followed by subacute sclerosing panencephalitis (SSPE), a fatal disease of Petition 870250085909, dated 09 / 23 / 2025, page 192 / 377 2 / 156 central nervous system involvement that typically develops 7 to 10 years after infection. Among people who contracted measles during the resurgence of the disease in the United States (US) between 1989 and 1991, the risk of SSPE was estimated at 7 to 11 cases / 100,000 measles cases. The risk of developing SSPE is higher when measles occurs before the age of two. The average incubation period for measles is 11 to 12 days, and the average interval between exposure and the onset of the rash is 14 days, with a range of 7 to 21 days. People with measles are generally considered infectious from four days before to four days after the onset of the rash, with the onset of the rash considered day zero.

[005] Before the introduction of the measles vaccine in 1963 and widespread vaccination, major epidemics occurred approximately every 2 to 3 years, and measles caused about 2.6 million deaths each year. More than 140,000 people died from measles in 2018 – most of them children under 5 years old, despite the availability of a safe and effective vaccine.

[006] The acceleration of immunization activities has had a major impact on reducing measles deaths. From 2000 to 2018, measles vaccination prevented approximately 23.2 million deaths. Measles deaths worldwide decreased by 73%, from an estimated 536,000 in 2000 to 142,000 in 2018. According to the World Health Organization (WHO), the largest measles outbreak was recorded in India in 2022, with 12,773 reported cases, making India's goal of eradicating measles by 2023 impractical. There was a recent measles outbreak in Maharashtra, with 3,075 cases and 13 deaths. According to the WHO, more than 58,000 people in 41 of the 53 Member States in the Region – spanning Europe and Central Asia – were infected with measles in 2023, resulting in thousands of hospitalizations and 10 measles-related deaths. Petition 870250085909, dated 09 / 23 / 2025, page 193 / 377 3 / 156

[007] Measles is still common in many developing countries – particularly in parts of Africa and Asia. The overwhelming majority (over 95%) of measles deaths occur in countries with low per capita income and poor health infrastructure. Measles outbreaks can be particularly deadly in countries experiencing or recovering from a natural disaster or conflict. Damage to infrastructure and health services disrupts routine immunization, and overcrowding in residential camps considerably increases the risk of infection.

[008] Mumps is an acute viral disease caused by a paramyxovirus that typically presents as swelling of the parotid gland (parotitis) or other salivary gland(s). Mumps occurs unilaterally or bilaterally and lasts 3 to 7 days (average 5 days); most cases resolve within 10 days. In some cases, nonspecific prodromal symptoms precede mumps 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 mumps usually develops 16 to 18 days after exposure to the mumps virus.

[009] Mumps can occur in fully vaccinated individuals, but vaccinated individuals have a much lower risk of developing mumps and its complications. Mumps reinfection can also occur in patients who have had a natural infection or recurrent mumps (the parotid swelling disappears and, weeks or months later, occurs on the same side or the other). Mumps infection may present only with nonspecific or primarily respiratory symptoms, or be asymptomatic. Among unvaccinated individuals, approximately 20% of infections are asymptomatic; the frequency of asymptomatic infection among vaccinated individuals is unknown. Petition 870250085909, dated 09 / 23 / 2025, pp. 194 / 377 4 / 156

[010] Worldwide, mumps is not as well controlled as measles and rubella. From 1999 to 2019, on average, about 500,000 cases of mumps were reported to the World Health Organization annually; however, the global incidence of mumps is difficult to estimate, as mumps is not a notifiable disease in many countries. In 2019, the mumps vaccine was routinely used in 122 of 194 (63%) countries. Since the mid-2000s, mumps outbreaks have also been reported among populations with high coverage of the two-dose MMR vaccine in other countries, including the UK, Ireland, New Zealand, Canada, the Netherlands, Spain, and Norway. Despite these outbreaks, the incidence of mumps is still much higher in countries that do not have routine mumps vaccination.

[011] Rubella is an acute, contagious viral infection that occurs most frequently in children and young adults. Rubella is the leading cause of vaccine-preventable birth defects. Although rubella virus infection usually causes mild fever and rash in children and adults, infection during pregnancy, especially during the first trimester, can result in miscarriage, fetal death, stillbirth, or in babies with congenital malformations, known as congenital rubella syndrome (CRS). The rubella virus is transmitted through airborne droplets when infected people sneeze or cough. Humans are the only known hosts. There is no specific treatment for rubella, but the disease can be prevented by vaccination.

[012] Rubella is a viral disease caused by a Togavirus of the genus Rubivirus and is characterized by a mild maculopapular rash. The rubella rash occurs in 80% of people infected with rubella and is sometimes misdiagnosed as measles or scarlet fever. Children usually develop Petition 870250085909, dated 09 / 23 / 2025, page 195 / 377 5 / 156 Few or no constitutional symptoms, but adults may present with a prodrome of 1 to 5 days with low-grade fever, headache, malaise, mild runny nose, and conjunctivitis. Retroauricular, 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 by direct contact or by droplets of nasopharyngeal secretions and has a median incubation period of 17 days (range: 12 to 23 days). People with rubella are most infectious when the rash is erupting, but may shed the virus 7 days before or 7 days after the onset of the rash.

[013] Measles and rubella vaccination is an integral part of immunization programs worldwide, contributing to progress toward achieving global immunization targets and, more broadly, the Global Health Security Agenda and the United Nations Sustainable Development Goals (SDGs).

[014] The Region of the Americas, which had eliminated measles in 2016, lost its disease elimination status in 2018. Globally, reported measles cases more than doubled from 2017 to 2018, rising from 170,000 to 350,000. This upward trend continued in 2019, with several countries facing major measles outbreaks. In 2019, the Democratic Republic of Congo, Ukraine, and Brazil reported 333,017, 57,282, and 18,203 confirmed measles cases, 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 for the first dose of the measles-containing vaccine (MCV1) below 50%, and 23 had coverage below 70%, indicating that 30 to 50% of children in these countries had not received any dose of the measles vaccine through service delivery mechanisms. Petition 870250085909, dated 09 / 23 / 2025, pp. 196 / 377 6 / 156 routine services.

[015] To achieve the goal of providing full vaccination coverage of over 90% and reaching the target of the universal immunization program, India launched the Intensified Indradhanush Mission (IMI) 4.0 in phases in 2022. Although measles and rubella prevention is a priority, according to FOGSI [Federation of Obstetric and Gynecological Societies of India (FOGSI)] recommendations, the MMR vaccine combination is preferable to the rubella vaccine for routine pre-conception vaccination purposes. Despite the availability of the MMR vaccine, recent measles outbreaks and the increased prevalence of mumps are alarming and justify broader MMR vaccination coverage in the country.

[016] The global market demand for MMR vaccines is approximately 110 million doses per year. Efficient vaccine production requires the cultivation of large quantities of viruses, produced in high yield from a host system. The process and cultivation conditions under which a viral strain is grown are of great importance in achieving an acceptable high yield of the strain. Therefore, to maximize the yield of the desired virus, both the system and the cultivation conditions must be specifically adapted to provide an advantageous environment for the production of the desired virus. Therefore, there is a continuous need for safe and effective methods to produce viruses and antigens.Furthermore, there is a need for an approach to viral propagation that employs readily available materials and requires minimal time-consuming manipulation, where selecting a combination of host cells, culture medium, growth conditions, and production system is essential to achieving an efficient production process.

[017] However, live attenuated vaccines are particularly Petition 870250085909, dated 09 / 23 / 2025, pp. 197 / 377 7 / 156 are fragile and their vaccine action is rapidly destroyed when exposed to temperatures above +4 °C and sometimes even lower. This instability of the vaccine is unacceptable, especially when these vaccines are used on a large scale in tropical countries, where they may be accidentally exposed to the high ambient temperatures prevalent in these countries. Therefore, it is necessary to have a stable and heat-resistant vaccine, capable of withstanding deterioration when stored for long periods and accidentally exposed to high temperatures.

[018] Vaccine stability depends on several factors, such as storage temperature, storage time, 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 at room or warm temperatures. Therefore, there is a need to develop vaccine composition methods that induce a protective immune response with improvements in vaccine stability and shelf life.

[019] There are several disadvantages associated with the use of whey and animal-derived components, mainly cost, batch-to-batch variability in their composition, their association with a higher risk of contamination by adventitious agents, and the subsequent difficulties encountered in further processing (e.g., purification to eliminate whey proteins or introduced animal-derived proteins).

[020] Before the start of the industrial-scale mammalian cell culture process in a bioreactor, a seed culture inoculum is typically prepared. This involves culturing production cells in a series of single or multiple plate flasks in incubators and / or smaller bioreactors of increasing volume until sufficient cells are available for inoculation into the bioreactor. Petition 870250085909, dated 09 / 23 / 2025, pp. 198 / 377 8 / 156 of production. The process involves transferring a population of cells 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 containing culture medium are fixed or attached to a rotating platform to agitate 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°C with 5% carbon dioxide (CO2) and a humidity level above approximately 80%. Similar temperatures and CO2 levels are used for seed cultures grown in bioreactors. When the seed culture reaches sufficient volume and cell density, it is inoculated into the production flask / bioreactor.

[021] After the seed culture is inoculated into the bioreactor medium, parameters such as pH, temperature, and dissolved oxygen level are controlled to the prescribed levels during the cell culture process. pH is typically controlled by adding basic or acidic solutions, as needed, during the process. Commonly used basic solutions include sodium bicarbonate, sodium carbonate, and sodium hydroxide solutions. Dissolving carbon dioxide (CO2) is commonly used to achieve a more acidic pH. Although other acids are available to control pH, the combination of dissolved CO2 and sodium bicarbonate forms a very stable and favorable buffer system for cell culture. The preferred temperature of the culture medium or solution for mammalian cell culture processes is approximately 37°C.The desired level of dissolved oxygen in the culture medium or solution is typically achieved by spraying air using a sprayer installed at the bottom of the bioreactor or through the headspace, along with agitation of the culture medium or solution using impellers that break up the culture medium. Petition 870250085909, dated 09 / 23 / 2025, page 199 / 377 9 / 156 Large air / oxygen bubbles are used to improve oxygen transfer to the cellular medium, either from sprayed air bubbles or using other gas exchange systems. Purging the bioreactor headspace with a blanket gas provides a limited degree of surface gas exchange. Disadvantages: Air spraying and agitation of the culture medium can result in foaming and shear damage to mammalian cells, negatively affecting cell viability. Foam buildup on the surface of the culture medium also further limits surface gas exchange and reduces the available working volume of the bioreactor.

[022] Mammalian cells are known to be sensitive to the amount of carbon dioxide dissolved in the cell culture medium. Mammalian cell cultures exposed to excessive levels of carbon dioxide during the exponential growth phase may exhibit reduced production of monoclonal antibodies or other desired biological products. Before inoculation, the pH of the slightly alkaline culture medium should be reduced with the carbon dioxide adjusted to an ideal value. This frequently 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 both chemical and biological sources.The chemical source of carbon dioxide is the equilibrium chemical reactions occurring within the cell culture medium, which includes a selected amount of a buffer solution containing sodium bicarbonate and / or sodium carbonate. Additionally, carbon dioxide can be sprayed directly onto the slightly alkaline culture medium to reduce the broth pH to a prescribed level, usually around 7.0, resulting in more dissolved carbon dioxide. The biological source of carbon dioxide is a product of mammalian cell respiration within the bior. Petition 870250085909, dated 09 / 23 / 2025, pp. 200 / 377 10 / 156 reactor. This biological source of carbon dioxide increases with cell density and generally reaches its maximum value at approximately the same time that the cell density within the bioreactor is maximized. However, as more carbon dioxide is produced, the pH of the cell culture medium tends to become acidic, so additional bicarbonate is needed to maintain the pH of the medium or cell culture solution within the desired range. To compensate for the effects of increased dissolved carbon dioxide, which depresses the pH, sodium bicarbonate can be added to maintain the pH of the solution within the prescribed range. Both ways of compensating for the effects of increased carbon dioxide have other negative consequences in the mammalian cell culture process.

[023] The addition of sodium bicarbonate, necessary to adjust the pH of the solution and compensate for carbon dioxide, also increases osmolality. (Osmolality represents the number of dissolved particles per kilogram of solution and is commonly reported as mOsm / kg per freezing point depression). The addition of sodium bicarbonate will also increase the equilibrium saturation level of dissolved carbon dioxide allowed in the solution, making the removal of carbon dioxide more difficult during the aeration process. It is known in the art that elevated levels of dissolved carbon dioxide or increased osmolality have adverse or negative impacts on cell density or yield.

[024] Carbon dioxide dissociates into bicarbonate ions at pH 7 in water. Only a fraction of the carbon dioxide remains as free CO2 in an undissociated state. Removing dissolved carbon dioxide from a cell culture is therefore difficult, since most mammalian cell cultures occur at pH in the range of 6.5 to 7.5. The dissociated bicarbonate ions are not easily Petition 870250085909, dated 09 / 23 / 2025, page 201 / 377 11 / 156 are removed and generally need to be recombined into free carbon dioxide before being removed from the solution.

[025] The conventional method for removing or removing dissolved carbon dioxide from a mammalian cell culture solution is by spraying the cell culture solution with air or a gaseous mixture of air / oxygen / nitrogen in stirred tanks. However, gas spraying in stirred tanks results in adverse effects on the cell culture process. In particular, gas bubble rupture at the tip of the rotary stirrer is a source of high shear rate that damages mammalian cell membranes, often enough to cause cell death. Even when the damage is sublethal, cell productivity is compromised during the period while the damaged membrane is repaired.

[026] Furthermore, the injection of air or nitrogen into the bioreactor creates gas bubbles that rise to the surface of the solution within the bioreactor, where the gas is released into the headspace. The rupture of the gas bubble on the upper surface of the cell culture solution is often more damaging to mammalian cells than the damage caused by the agitator. Restricting the agitator speed and limiting the gas injection rate are currently considered 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 difficult to overcome in large-scale commercial bioreactors, where the shear rate increases substantially with the impeller diameter.Furthermore, the higher hydrostatic pressure of large-scale bioreactors tends to increase the solubility of carbon dioxide, meaning that more carbon dioxide needs to be removed to keep dissolved CO2 levels within a certain range. Petition 870250085909, dated 09 / 23 / 2025, page 202 / 377 12 / 156 ideal.

[027] According to the parameters explained above, a manufacturing process is needed that can overcome the problems related to the process parameters / steps involved in the manufacture of the measles vaccine.

[028] Consequently, there is a need for a method of large-scale, economical and safe production of viral vaccines that includes a minimum of components of animal origin, provides high viral yield and guarantees the integrity / stability of the viral structure during manufacturing and storage.

[029] The applicant surprisingly found large-scale, accessible and safe manufacturing processes (covering the cultivation, purification and formulation steps) that use a minimum of animal-derived components, provide high viral yield and ensure the integrity / stability of the viral structure during manufacturing and storage, in which 1) recombinant trypsin is used (at least 95% purity, with a molecular weight of 23 to 25 kilodaltons; specific activity of 2500 USP units / mg; low endotoxin content) at an ideal pH (7.2 to 7.6); 2) Obtaining viral yield > 50% (increase of 30 to 40%) due to ideal pH (7.2 to 7.8), ideal concentration of sodium bicarbonate in the medium (1.0 to 2.5 g / L), low multiplicity of infection (MOI), i.e., virus / cell ratio (for mass production of measles and rubella vaccines - 1:5 to 1:20 and for mass production of mumps vaccines - 1:20 to 1:60), multiple sampling;3) Use of FBS irradiated with gamma rays (dose range of 20 to 50 kgy); 4) Ideal virus:stabilizer-I:stabilizer-II ratio (80:20:10); 5) Use of vented MLTCF-10 with Corning vented caps (instead of Milex filter), resulting in slow dissociation of sodium bicarbonate. OBJECTIVES Petition 870250085909, dated 09 / 23 / 2025, page 203 / 377 13 / 156

[030] Some of the objectives of the present description, which at least one modality described here satisfies, are the following:

[031] One objective of the present description is to alleviate one or more problems of the prior art or, at least, to provide a useful alternative.

[032] One objective of the present description is to provide a pool of clarified viruses in the manufacture of freeze-dried / freeze-dried live attenuated virus vaccine that includes one or more viruses.

[033] Another objective of the present description is to provide an improved method for manufacturing a live attenuated lyophilized viral vaccine composition.

[034] Another objective of the present description is to provide an improved method for manufacturing a live attenuated lyophilized viral vaccine composition comprising at least one virus selected from a group consisting of measles, mumps and rubella.

[035] Another objective of the present description is to provide an easy and simple method for the mass production of live attenuated viral vaccine, which may contribute to increasing the final yield of mass-produced vaccine and doses.

[036] Another objective of the present description is to provide an easy and simple method for the manufacture of a live attenuated lyophilized viral vaccine composition that can contribute to increasing the final yield of mass-produced vaccine and doses.

[037] Yet another objective of the present description is to provide a method for manufacturing a live attenuated lyophilized viral vaccine composition using gamma-ray irradiated FBS.

[038] Yet another objective of the present description is to provide a method for manufacturing a live attenuated lyophilized viral vaccine composition using recombinant trypsin at optimum pH.

[039] Yet another objective of the present description is to provide a Petition 870250085909, dated 09 / 23 / 2025, page 204 / 377 14 / 156 method for manufacturing a live attenuated lyophilized viral vaccine composition with high viral yield using optimum pH, optimum sodium bicarbonate concentration in medium, low MOI ratio, i.e., virus / cell, and multiple sampling.

[040] Yet another objective of the present description is to provide a method for manufacturing a live attenuated lyophilized viral vaccine composition using vented MLTCF-10 with Corning vented caps (instead of Milex filter).

[041] Yet another objective of the present description is to provide a live attenuated lyophilized viral vaccine composition containing the ideal virus:stabilizer ratio.

[042] Another objective of the present description is to provide a live attenuated / lyophilized lyophilized viral vaccine composition / formulation in which, after reconstitution, the composition preserves the desired characteristics of the virus, including stability and immunogenicity.

[043] Other objectives and advantages of the present description will become more evident from the description that follows, which is not intended to limit the scope of the present description. SUMMARY

[044] The applicant provides a method for producing a clarified virus pool, the method comprising: providing a cell line in a cell medium and supplement; treating cells of the cell line with enzyme; infecting the cell line with a virus to form an infected cell line; washing the infected cell line with the viral medium; harvesting the infected cell line in the medium to obtain a harvest; optionally, collecting the infected cell line; adding a stabilizer to the harvest; and clarifying the harvest to obtain a clarified virus pool (CVP).

[045] In one aspect, the present invention relates to a method for producing a clarified viral pool, comprising the method: Petition 870250085909, dated 09 / 23 / 2025, page 205 / 377 15 / 156 a. provide a cell line in a cell medium, a buffer, and a supplement; b. treat cells from the cell line with at least one enzyme; c. infect the cell line with a virus to form an infected cell line; d. Wash the infected cell line with a viral medium and buffer; e. harvest the infected cell line from the medium to obtain a harvest; optionally, collect the infected cell line one or more times; f. add at least one stabilizer to the harvest; and g. clarify the sample to obtain a clarified viral pool (CVP). optionally, where the cell line or the infected cell line in the exponential growth phase is - grown with additional ventilation or aeration, or - provided with tampon readmission or both.

[046] In another aspect, the present invention relates to a clarified viral pool (CVP) obtained by the method described.

[047] In another aspect, the present invention relates to a method for producing a pool of clarified measles virus.

[048] In another aspect, the present invention relates to a pool of clarified measles virus (CVP) obtained by the method described.

[049] In another aspect, the present invention relates to a method for producing a pool of clarified mumps virus.

[050] In another aspect, the present invention relates to a pool of clarified mumps virus (CVP) obtained by the method described.

[051] In another aspect, the present invention relates to a mé Petition 870250085909, dated 09 / 23 / 2025, pp. 206 / 377 16 / 156 all to produce a pool of clarified viruses for rubella.

[052] In another aspect, the present invention relates to a pool of clarified rubella virus (CVP) obtained by the method described.

[053] In another aspect, the present invention relates to a method for obtaining a freeze-dried / lyophilized immunogenic composition for measles, mumps and rubella (MMR), comprising one or more CVPs selected from a CVP for measles, a CVP for mumps, a CVP for rubella or a combination thereof, mixing the CVPs, followed by freeze-drying the combined CVP.

[054] In another aspect, the present invention relates to a freeze-dried / lyophilized immunogenic composition for MMR.

[055] In another aspect, the present invention relates to a kit comprising the lyophilized / freeze-dried immunogenic composition for MMR.

[056] The applicant provides a method for producing a pool of clarified viruses, such as measles, mumps, and rubella. The enzymatic treatment performed on the cell line is with recombinant trypsin. The washing of the infected cell line is performed with FBS-free viral medium. The buffer added to the cell medium and viral medium is selected NaHCO3 (sodium bicarbonate). Sodium bicarbonate in viral medium maintains physiological pH, which provides significant results. The applicant has found improved, accessible, and safe large-scale manufacturing processes (encompassing the cultivation, purification, and formulation steps) that use minimal animal-derived components, provide high viral yield, and ensure the preservation of viral structure integrity / stability during manufacturing and storage, in which: 1) Recombinant trypsin (at least 95% pure, with a molecular weight of 23 to 25 Petition 870250085909, dated 09 / 23 / 2025, pp. 207 / 377 1) 17 / 156 kilodaltons; specific activity of 2500 USP units / mg; low endotoxin content) at ideal pH (7.2 to 7.6); 2) Obtaining viral yield > 50% (increase of 30 to 40%) due to ideal pH (7.2 to 7.8), ideal concentration of sodium bicarbonate in the medium (1.0 to 2.5 g / L), low MOI, i.e., virus / cell ratio (for mass production of measles and rubella vaccines - 1:5 to 1:20 and for mass production of mumps vaccines - 1:20 to 1:60), multiple harvesting; 3) use of FBS irradiated with gamma rays (dose range of 20 to 50 kgy); 4) ideal virus:stabilizer-I:stabilizer-II ratio (80:20:10); 5) Use of vented MLTCF-10 with Corning vented caps (instead of Milex filter), resulting in slow dissociation of sodium bicarbonate. DETAILED DESCRIPTION OF THE DRAWING

[057] The present invention will now be described with the aid of the accompanying drawing, in which: Figure 1 illustrates the production of the measles virus. Figure 2 illustrates the buffer dissociation equation. DEMMRITION

[058] Although the present description may be susceptible to different embodiments, certain embodiments are shown in the drawing and detailed discussion below, it being understood that the present description may be considered an exemplification of the principles of description and is not intended to limit the scope of description to what is illustrated and described in this description.

[059] The embodiments are provided in such a way as to convey the scope of the present description completely and thoroughly to a person skilled in the art. Numerous details are presented, relating to specific components and processes, to provide a complete understanding of the embodiments of the present description. It will be evident to a person skilled in the art that the details provided Petition 870250085909, dated 09 / 23 / 2025, pp. 208 / 377 18 / 156 in the modalities should not be interpreted as limiting the scope of the present description. In some modalities, well-known compositions, processes and techniques are not described in detail.

[060] The terminology used in this description serves only to explain a specific modality and should not be considered as limiting the scope of this description. As used in this description, the forms “um”, “uma” and “o(a)” may also include the plural, unless the context clearly suggests otherwise.

[061] The terms “comprises”, “comprising”, “including” and “having” are open transition phrases and therefore specify the presence of the declared features, integers, steps, operations, elements, modules, units and / or components, but do not prohibit the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof. The specific order of the steps described in the process of this description should not be interpreted as necessarily requiring their execution as described or illustrated. It should also be understood that additional or alternative steps may be employed.

[062] The terms first, second, third, etc., should not be interpreted as limiting the scope of this description, since the aforementioned terms may only be 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 this description.

[063] It is understood that each characteristic or modality, or combination, described here is an illustrative and not limiting example of Petition 870250085909, dated 09 / 23 / 2025, page 209 / 377 19 / 156 any aspect of the invention and, as such, must be combinable with any other feature or embodiment, or combination, described herein. For example, when features are described with terms such as “an embodiment”, “some embodiments”, “certain embodiments”, “additional 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 the need to list all possible combinations. Such features or combinations of features apply to any aspect of the invention. Definitions:

[064] To make this description easier to understand, certain terms are first defined below. Additional definitions for the following terms and other terms may be presented throughout the specification.

[065] The term “PDL” stands for “population doubling level” and refers to the total number of times cells in a given population have doubled during in vitro culture.

[066] The term “working cell bank” refers to the quantity of cells of uniform compositions derived from the master cell bank at a finite throughput level, dispensed in aliquots into individual containers suitably stored in liquid nitrogen containers at -196 °C, one or more of which would be used for production purposes.

[067] The term “cellular medium” can be used interchangeably with the term “cellular growth medium”.

[068] The term “viral medium” can be used interchangeably with the terms “viral growth medium” and “washing medium”.

[069] The terms “CF” refer to “cell factory” and “CS” refers to Petition 870250085909, dated 09 / 23 / 2025, p. 210 / 377 20 / 156 are injured by the "cell stack".

[070] The terms MLTCF refer to “multilayer tissue culture flask”.

[071] The term “working seed virus” refers to the virus used to infect the cell line.

[072] The term “lyophilization / freeze drying / lyophilization / freeze drying” 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.

[073] The term “sublimation” refers to a change in the physical properties of a composition, in which the composition changes directly from the solid state to the gaseous state without becoming liquid.

[074] The term “Tresivac” refers to the “Live Attenuated (Lyophilized) Measles, Mumps and Rubella Vaccine (MMR vaccine) of the Serum Institute of India, Ltd.”

[075] The term “Blind Vaccine” refers to the diluent used for dilution of bulk virus or pool(s) of virus clarified during mixing, or used for mixing with bulk virus or pool(s) of virus clarified during mixing.

[076] The term “mass vaccine” refers to the solution containing the cleared virus pool of viruses or the mixed solution containing the cleared virus pool of more than one virus.

[077] The term “mass vaccination” can be used interchangeably with the term “mass virus vaccination”.

[078] The term “dose” here typically refers to one administration of the vaccine of the invention, which is normally an injection. A typical human dose is 0.5 mL. Of course, multiple doses may be administered in a vaccine administration schedule.

[079] The present description provides a method for the manufacture of viral vaccines. In a preferred embodiment, the present description Petition 870250085909, dated 09 / 23 / 2025, p. 211 / 377 21 / 156 provides a method for manufacturing a freeze-dried / freeze-attenuated live virus vaccine that includes one or more viruses. The freeze-dried / freeze-attenuated virus vaccine includes the majority of one or more vaccine viruses.

[080] The present description provides a method for producing a pool of clarified viruses in the manufacture of a freeze-dried / lyophilized live attenuated virus vaccine that includes one or more viruses.

[081] In a preferred embodiment, the present description provides a method for producing a pool of clarified viruses in the manufacture of a freeze-dried / lyophilized live attenuated virus vaccine that includes one or more viruses and increases the viral titer by the addition of media in the manufacturing process.

[082] In a preferred embodiment, the present description provides a method for producing a pool of clarified viruses in the manufacture of a freeze-dried / lyophilized live attenuated virus vaccine that includes one or more viruses and increases the viral titer by adding post-infection virus media in the manufacturing process.

[083] In a preferred embodiment, the present description provides a method for producing a pool of clarified virus in the manufacture of a freeze-dried / lyophilized live attenuated virus vaccine that includes one or more viruses, increasing viral production by adding media after infection with the virus in the manufacturing process.

[084] In a preferred embodiment, the present description provides a method for producing a pool of clarified virus in the manufacture of a freeze-dried / lyophilized live attenuated virus vaccine that includes one or more viruses, increasing the viral load by adding media after infection with the virus in the manufacturing process.

[085] In a preferred embodiment, the present description provides Petition 870250085909, dated 09 / 23 / 2025, page 212 / 377 22 / 156 is a method for producing a pool of clarified viruses in the manufacture of a freeze-dried / lyophilized live attenuated virus vaccine that includes one or more viruses, increasing the final mass production of the vaccine and doses by adding media after infection with the virus in the manufacturing process.

[086] In a preferred embodiment, the present description provides a method for producing a pool of clarified virus in the manufacture of freeze-dried / freeze-dried live attenuated virus vaccine that includes one or more viruses, increasing viral titer, viral yield, final mass vaccine yield and doses by adding means after infection with the virus in the manufacturing process. GENERAL METHOD FOR PRODUCING CVP

[087] In one aspect of the present description, the method according to the present description comprises: a. provide a cell line in a cell medium, a buffer, and a supplement; b. treat the cells of the cell line with at least one enzyme; c. infect the cell line with a virus to form an infected cell line; d. Wash the infected cell line with a viral medium and buffer; e. harvest the infected cell line from the medium to obtain a harvest; optionally, collect the infected cell line; f. add at least one stabilizer to the harvest; and g. clarify the harvest to obtain a clarified virus pool (CVP). Optionally, where the cell line or the infected cell line in the exponential growth phase is: Petition 870250085909, dated 09 / 23 / 2025, page 213 / 377 23 / 156 - grown with additional ventilation or aeration, or - supplied with additional buffer or - both.

[088] In one embodiment, the CVP production method is for the mass preparation of a viral vaccine / mass viral immunogenic composition.

[089] In another modality, the method for producing CVP includes: a) To provide a virus-dependent cell line; b) Reactivate and transfer cells into a tissue culture flask (TCF); c) Preparing cell factories / cell stacks; d) Treating cell lines with at least one enzyme, the enzyme including trypsin, i.e., trypsinization of cell factories / cell stacks; e) Infecting the cell line by adding viruses, that is, infection; f) Wash the infected cell line using viral medium and buffer; g) Harvest the infected cell line from the medium to obtain the harvest; harvesting of infected cell lines to obtain multiple harvests; h) Add at least one stabilizer to the sample; i) Clarify the sample to obtain the clarified viral pool (CVP) j) Store the CVP below -20°C as a pharmaceutical substance, i.e., mass viral vaccine, optionally, where the cell line or the infected cell line is in the exponential growth phase: - grown with additional ventilation or aeration, or Petition 870250085909, dated 09 / 23 / 2025, page 214 / 377 24 / 156 - supplied with additional buffer or - both. STEP a):

[090] In one embodiment, the method of producing the clarified viral pool includes supplying the cell line in the cell medium, buffer and supplement.

[091] In one embodiment, the cell line is provided in a container. The container includes covered flasks, bottles, rolling flasks, serrated rolling flasks, cuboid containers, round-bottom containers, and cell factories.

[092] In one embodiment, the container is covered. The covering of the container includes lids. CELL LINE

[093] In another embodiment, the cell line used as a substrate for virus growth includes animal cell line, insect cell line, human cell line, primary cell line (chicken embryo fibroblast (CEF)), diploid cell line (including human lung fibroblast (MRC-5)) and continuous cell line.

[094] In one embodiment, the cell line used as a substrate for virus growth is selected from among different cell lines, such as Rhesus monkey kidney cells (RhMK); rabbit primary kidney cells; human prepuce fibroblasts; chicken embryo fibroblasts (CEF); human squamous cell carcinoma cells (HEp-2); human lung carcinoma cells (A549); human cervical epithelial cells (HeLa); African green monkey kidney epithelial cells (Vero); human lung fibroblasts (MRC-5); human lung fibroblasts (MRC-9); mouse embryo fibroblasts (NIH3T3); mouse connective tissue fibroblast (L929); hamster ovary fibroblast Petition 870250085909, dated 09 / 23 / 2025, pp. 215 / 377 25 / 156 nês (CHO); Syrian hamster kidney fibroblast (BHK-21); Human embryonic renal epithelial (HEK-293); Human liver epithelial (HepG2); Bovine aortic endothelial (BAE-1); Human neuronal neuroblastoma (SH-SY5Y); Mouse myeloma lymphoblast (NSO); Human histiocytic 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); Canine Madin Darby kidney cells (MDCK); human embryonic retinal cells (PER.C6); human embryonic retinoblasts (HER.911); murine non-secretory myeloma (Sp2,0); African green monkey kidney epithelial cells (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 accelerating 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); swine kidney cells (PK15 cells); rabbit kidney cell line (LLC-RK1 cells); non-secretory myeloma cell lines (NS-1 cells); novel human male diploid cell line (TIG-1, TIG-7); non-human primate diploid cell line (FRhL-2); human fetal lung cells (IMR-90, IMR-91); Human diploid lung fibroblasts (including WI-38) and others.

[095] In one embodiment, the cell line used as a substrate for the growth of measles, mumps, and rubella viruses is selected from the lung fibroblast cell line. Petition 870250085909, dated 09 / 23 / 2025, pp. 216 / 377 26 / 156 humans (MRC-5) and the chicken embryo fibroblast cell line (CEF).

[096] In another embodiment, MRC-5 cells in lower passage were obtained from NIBSC, UK. Master and working cell banks are prepared and stored at -196 °C in liquid nitrogen. This cell stock is used for production purposes. The cell stock was characterized and tested according to Ph. Eur and WHO.

[097] In another embodiment, chicken embryo fibroblast cells (CEF) are prepared using 9- to 11-day-old embryos from specific pathogen-free (SPF) chicken eggs. These SPF eggs are received from Lohmann Germany and Hy-Vac, USA, etc.

[098] In one embodiment, the cell line is provided with the cell medium, the supplement and the buffer. CELLULAR MEDIUM

[099] In one embodiment, for the CVP production method, the cell line is provided with a cell medium (hereinafter referred to interchangeably as cell growth medium) for growth and propagation. The cell medium contains nutrients, including carbon sources, carbohydrates, vitamins, amino acids, minerals, growth factors, hormones, sugars, glucagon, cyclodextrin, inorganic salts, buffers, or a combination thereof.

[0100] The carbon source includes carbohydrates, glucose, glutamine, sucrose, dextrose, galactose, fructose, or combinations thereof.

[0101] In another embodiment, the cell growth medium is selected from basal medium, enriched medium, selective and indicator medium, transport medium and storage medium.

[0102] In another embodiment, the cell growth medium is selected from among different media, such as basal culture media, cell culture media suitable for the growth of a wide range of species Petition 870250085909, dated 09 / 23 / 2025, pp. 217 / 377 27 / 156 mammalian cell culture, Minimum Essential Medium supplemented with L-Glutamine, multipurpose media, minimum essential medium modified to contain Balanced Saline Solution (BSS) and amino acids.

[0103] In another embodiment, balanced saline solution includes Earle's Balanced Saline Solution (EBSS), Gey's Balanced Saline Solution (GBSS), Hanks' Balanced Saline Solution (HBSS), Dulbecco's Phosphate Buffered Saline Solution (PBS), Puck's Balanced Saline Solution, Ringer's Balanced Saline Solution (RBSS), Alsever's Solution, Simm's Balanced Saline Solution (SBSS), TRIS Buffered Saline Solution (TBS), Tyrode's Balanced Saline Solution (TBSS). In a preferred embodiment, the Balanced Saline Solution is Earle's or Hanks' BSS.

[0104] In one embodiment, the cell growth medium is selected from Medium 199 (M-199), Dulbecco Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM); Eagle Minimum Essential Medium (E-MEM), Hank's MEM (H-MEM), Iscove Modified Dulbecco Medium (IMDM), Ham's nutrient mixtures (F-10 and F12), Leibovitz (L-15), Roswell Park Memorial Institute (RPMI)-1640; Neurobasal Medium; Schneider's Drosophila Medium; McCoy's 5A Medium; Dynamis Medium; Essential Medium 8 (E8); StemFlex culture medium; Basal medium of airway epithelial cells; Alpha-modified minimum essential medium (α-MEM); StemMacs iPS-Brew medium; TeSR-E8, mTeSRI, mTeSR Plus medium; GMEM (Glasgow Minimum Essential Medium); Opti-MEM I; SmGM-2; fibroblast growth medium / FGM; serum-free growth medium StemPro-34; mTESRI medium; ECGM-2 medium; Williams E medium; M254 medium; CnT07 medium; TNMFH medium; basal medium for mammary epithelial cell growth (MEBM);Medium for complete skeletal muscle and basal medium NeuroCult NS-A.;

[0105] In another modality, the cell growth medium as Petition 870250085909, dated 09 / 23 / 2025, pp. 218 / 377 28 / 156 synthetic cell culture medium is the Minimum Essential Medium. The medium is sterilized using sterile-grade hydrophilic filters.

[0106] In a preferred embodiment, filtration of the medium is carried out using sterile grade hydrophilic filters of 0.1 pm.

[0107] In another embodiment, the cell growth medium is supported or supplemented with buffer and supplements.

[0108] In another embodiment, the cell growth medium is Minimum Essential Medium (MEM), prepared using powdered MEM supplemented with L-Glutamine, fetal bovine serum (FBS) and pH buffer, mixing all ingredients in pre-sterilized water for injection. The medium is sterilized by 0.1 μ filtration. BUMP

[0109] In one embodiment, the buffer is used for the CVP production method.

[0110] The buffer includes NaHCO3, NaOH, NaCl, (4-(2-hydroxyethyl)-1-piperazinoethanesulfonic acid), i.e., HEPES, (piperazino-N,N'-bis(2-ethanesulfonic acid)), i.e., PIPES, or (2-(N-morpholinojetanesulfonic acid), i.e., MES.

[0111] In one embodiment, the buffer includes NaHCO3 (sodium bicarbonate).

[0112] In a preferred embodiment, the buffer, i.e., NaHCO3 (sodium bicarbonate), is used in a batch in the concentration range of 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.

[0113] In a more preferable embodiment, the buffer, i.e., NaHCO3 (sodium bicarbonate), is used in a batch in the concentration range of 1.0 to 1.9 g / L, or 1.2 to 1.9 g / L.

[0114] In another embodiment, the infected cell line is washed with viral medium and buffer. In another embodiment, the cell line or the infected cell line in the exponential growth phase is Petition 870250085909, dated 09 / 23 / 2025, page 219 / 377 29 / 156 grown with the addition of the buffer.

[0115] In another embodiment, the cell line or the infected cell line in the exponential growth phase is cultured with additional ventilation or aeration and readdition of the buffer.

[0116] In another embodiment, the readdition of the buffer is carried out by reinforcement with additional buffer, i.e., NaHCOs, or by supplying an additional quantity of the buffer with the cellular medium or the viral medium, or both.

[0117] In one embodiment, the concentration of the enhanced buffer for the cellular medium or the viral medium, or both, during the readdition of the buffer, is in the range of 5% to 40%, or 5% to 35%, or 5% to 30%.

[0118] In one embodiment, the concentration of the enhanced buffer for the cellular medium or the viral medium, or both, during the readdition of the buffer, is in the range of 10% to 30%, or 15% to 30%.

[0119] In one embodiment, the concentration of the enhanced buffer for the cellular medium or the viral medium, or both, during the readdition 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.

[0120] In one embodiment, the total buffer concentration, including the enhanced buffer concentration for the cellular medium or the viral medium, 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 batch.

[0121] In another embodiment, the total buffer concentration for the cellular medium or the viral medium, or both, including the enhanced 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 batch.

[0122] In a preferred embodiment, the total buffer concentration for the cellular medium or the viral medium, or both, including the enhanced buffer concentration, is in the range of 1.5 g / L to 2.0 g / L in Petition 870250085909, dated 09 / 23 / 2025, pp. 220 / 377 30 / 156 batch.

[0123] In one embodiment, the CVP preparation method includes providing the cell line cells with cell medium, buffer, and at least one supplement.

[0124] pH is a critical process parameter for cell growth. pH is controlled by adequate ventilation to allow gas exchange and by increasing the buffering capacity of the cell medium by adding an additional amount of buffer to the cell medium, which does not cause pH fluctuation, increases cell count and increases yield.

[0125] Therefore, pH is one of the critical parameters in the viral vaccine manufacturing process. Exposure of the virus to pH extremes inactivates it. The virus has been reported to be stable at pH in the range of 6 to 8, with progressive inactivation on both sides. The physical stability of the virus is highly compromised in an acidic environment. Even small changes in pH affect the native confirmation of the virus.

[0126] pH regulation is particularly important immediately after cell seeding, when a new culture is establishing itself, and is generally achieved by one of two buffering systems: (i) a “natural” buffering system, where gaseous CO2 equilibrates with the 0 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 to 10% CO2 in the air, usually provided in a CO2 incubator. The bicarbonate / CO2 buffering system is low-cost, non-toxic, and also provides other chemical benefits to the cells. HEPES has superior buffering capacity in the pH range of 7.2 to 7.4, but is relatively expensive and can be toxic to some cell types at higher concentrations. Cultures buffered with HEPES do not re Petition 870250085909, dated 09 / 23 / 2025, pp. 221 / 377 31 / 156 want a controlled gaseous atmosphere.

[0127] In one embodiment, cell growth is controlled by adding an additional amount of NaHCO3 to the cell medium, which does not cause pH fluctuation, increases cell count, and increases yield. pH, as one of the critical process parameters for cell growth, is controlled by adequate ventilation to allow gas exchange and increase the buffering capacity of the cell medium, adding an additional amount of NaHCO3 to the cell medium, which does not cause pH fluctuation, increases cell count, and increases yield. SUPPLEMENTS

[0128] In one embodiment, 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, adhesion and extension factors.

[0129] In another form, at least one supplement includes serum, antibiotics, and additional amino acids.

[0130] In another form, at least one supplement includes serum and antibiotics.

[0131] In another embodiment, at least one supplement includes antibiotics and additional amino acids. In another embodiment, at least one supplement includes serum and additional amino acids.

[0132] In one embodiment, the serum as a supplement is bovine serum or calf serum. The serum as a supplement is fetal bovine serum or fetal calf serum. The serum as a supplement is fetal bovine serum.

[0133] Fetal bovine serum (FBS) is the most widely used basal medium supplement for in vitro cell culture. It contains very low levels of antibodies and a high concentration of growth factors, such as hormones, binding factors, and trans proteins. Petition 870250085909, dated 09 / 23 / 2025, pp. 222 / 377 32 / 156 size. FBS is used to keep cells alive for a longer period. FBS contains a large number of components, such as growth factors, proteins, vitamins, trace elements, and hormones, which are essential for cell growth and maintenance. FBS effectively promotes and sustains cell growth at the low densities used for biological manufacturing. Serum also adds buffering capacity to the medium and binds or neutralizes toxic components. FBS is obtained from fetuses harvested from healthy animal slaughterhouses. The manufacturer's serum batches contain pools of serum collected from various animals.

[0134] Because it is a material of animal origin, FBS presents an inherent risk of contamination with transmissible adventitious agents. Bovine serum can be contaminated by various bovine viruses. It is evident that each batch of serum should be tested for ubiquitous and known-risk viruses. The possibility of introduction and replication of adventitious agents during cell culture has long been recognized as a potential risk leading to contamination of the final product by viruses. There have been several cases where laboratory studies have provided evidence of the presence of adventitious agents in marketed vaccines. These risks of adventitious agents are normally mitigated by analysis for the absence of adventitious agents in bovine serum. Most regulatory bodies permit the use of animal-derived materials only when their use can be justified due to the absence of viable alternatives.For this purpose, each batch of serum must be tested for sterility and adventitious agents. Virus testing is typically performed in accordance with various regulatory guidelines. However, testing methods have their own limitations, and sometimes the contaminating adventitious agent may not be detected.

[0135] The SFB undergoes post-manufacturing treatment methods of Petition 870250085909, dated 09 / 23 / 2025, page 223 / 377 33 / 156 as follows: 1. Filtration: Triple filtration at 0.1 µm has become the standard aseptic processing method for various types of serum. While this methodology removes bacteria and most mollicutes, it cannot render the serum free of all viruses. Nanofiltration using viral-retaining filters (nominal pore size of 20 nm) can be effective in removing even small viruses such as parvovirus. However, it suffers from scalability issues and flow degradation with serum materials. Therefore, nanofiltration is not suitable for large-scale processing of serum products. 2. UV Treatment: Ultraviolet irradiation (typically 254 nm) has proven effective in reducing pathogens. The method is not only effective against larger microbes, such as bacteria and mollicutes, but is also very effective against most viruses. It is commonly used for disinfecting large volumes of water. UV irradiation has also demonstrated effectiveness in small-scale experiments aimed at removing adventitious agents from cell culture media. However, it has not yet been optimized to effectively handle the large volumes of media required for commercial-scale bioproduction, and further technological development is needed. 3. High-Temperature Short-Time Treatment (HTST): Effective viral load reduction has been demonstrated for most viruses following HTST treatment. More heat-resistant viruses, such as parvoviruses, require temperatures above 115°C. A significant problem with this modality is that the biological activity of the serum can be seriously compromised at the relatively high temperatures required for treatment. This can result in significantly reduced cell growth in several cell lines. Petition 870250085909, dated 09 / 23 / 2025, page 224 / 377 34 / 156 4. Traditional Thermal Inactivation: Routine heat treatment of animal serum is performed in many cell culture laboratories as one of their standard procedures and is included in many biofabrication protocols. A wide temperature range, from 45 to 62 °C, and times of 15 to 60 minutes may be required. The most common methodology requires heating the serum to 56 °C for 30 minutes. Variability has been identified in: (1) exact temperatures and exposure times used; (2) mixing of the serum in the flasks; and (3) depth of water in the water bath relative to the height of the serum flasks. These factors can negatively impact the biological activity of the serum after treatment. For these reasons, thermal inactivation of FBS is not a recommended practice by ISIA unless its necessity for the specific cell culture application has been proven. 5. Chemical Treatment: Whey treatments with chemicals such as ethyleneimine or betapropiolactone (BPL) have also been used as strategies to mitigate the risk of adventitious agents in specific applications. Chemical treatment processes present specific risks that must be managed to ensure operator safety and are not commonly used commercially on a large scale. Ozone technology (whey ozonation) is not suitable for whey treatment as it causes severe oxidation / peroxidation of whey components essential for cell culture growth, significantly reducing whey performance. 6. Ionizing radiation: The various forms of ionizing radiation with the potential to reduce the microbial and viral load in serum include: electron beam and X-ray irradiation. a. The electron beam uses high-energy electrons to inactivate contaminants. However, the electron beam Petition 870250085909, dated 09 / 23 / 2025, page 225 / 377 35 / 156 does not have the necessary penetration capacity to irradiate large pre-filled vials (e.g., 500 mL and 1 L plastic vials) of serum. b. X-ray irradiation should theoretically combine the best characteristics of electron beam and gamma irradiation. However, the commercial application of X-ray irradiation for this purpose has been limited due to a lack of facilities capable of handling large batches of serum.

[0136] In one embodiment, fetal bovine serum as a supplement is treated with gamma irradiation. Gamma irradiation is one of the most widely used and effective methods for the inactivation of viruses and mycoplasmas in animal sera. Gamma irradiation possesses both the penetrating power and ease of handling that allow its routine use for pathogen reduction in ready-to-use serum bottles. Gamma irradiation is a very efficient and direct means of inactivating many different types of viruses in FBS. Because FBS can be contaminated by adventitious viruses, gamma irradiation is, after routine quality control for virus detection, the best method to increase the safety of its use in the production of biological products. Gamma irradiation is performed on the serum in the original product packaging. It is usually performed at low temperatures, thus maintaining the quality and performance of the serum unaltered.

[0137] In one embodiment, FBS is irradiated with gamma radiation in the dose range of 20 to 50 kgy. The gamma-irradiated FBS is used as a supplement in the cell culture medium. The gamma-irradiated FBS is not used as a supplement in the viral culture medium. This results in a substantial saving of FBS and results in a more cost-effective step.

[0138] Fetal bovine serum (FBS) is used during the formation of the clarified viral pool, as well as in the preparation of the viral vaccine in Petition 870250085909, dated 09 / 23 / 2025, pp. 226 / 377 36 / 156 mass.

[0139] In one embodiment, FBS is provided in the range of 5% to 15%, preferably 10% to 12.5% ​​of the batch.

[0140] In one embodiment, the supplemental antibiotic is not added at any stage of cell or viral growth. In one embodiment, the supplemental antibiotic is added at any stage of cell or viral growth. In one embodiment, the supplemental antibiotic is selected from the group consisting of kanamycin, streptomycin, and neomycin; preferably neomycin. Antibiotics are commonly used in cell culture to prevent contamination, maintain aseptic conditions, or select cells containing genetic modifications.

[0141] In one embodiment, the additional amino acid as a supplement includes glutamine. STEP b):

[0142] In one embodiment, the method of producing the clarified viral pool includes treating the cell line cells with at least one enzyme. ENZYME

[0143] In one embodiment, for the CVP production method, the cells of the cell line are treated with at least one enzyme.

[0144] In one embodiment, the enzyme is selected from trypsin, recombinant trypsin, dipase, collagenase, hyaluronidase, elastase, cysteine ​​protease, deoxyribonuclease I, and chymotrypsin.

[0145] In a preferred embodiment, the enzyme includes trypsin.

[0146] Trypsin is a proteolytic enzyme (serine protease) used as a cell dissociating agent in tissue culture techniques. As such, it becomes a critical raw material for all tissue culture-based vaccines, such as measles, mumps, rubella. Petition 870250085909, dated 09 / 23 / 2025, pp. 227 / 377 37 / 156 IA, rabies, rotavirus, etc.

[0147] Trypsinization is an essential technique to be considered for optimizing the MRC-5 cell culture process. For a vaccine production process, trypsinization can result in additional medium costs and increased contamination risks. On the other hand, very high trypsin activities in the virus production medium can result in cell detachment. Therefore, optimizing trypsin activity is essential.

[0148] In particular, variations in trypsin between batches, culture media, and differences in shear stress of culture systems need to be considered. Increased trypsin activity can result in an early increase in hemagglutinin (HA) titer in serum-free media, while very low concentrations may prevent successful viral replication. Furthermore, increased trypsin activity can complicate large-scale production processes; therefore, a single addition of trypsin may be preferable.

[0149] Porcine trypsin is used as a cell dissociation reagent in tissue culture technology. These preparations are nothing more than acetone extracts of porcine pancreas and typically contain a mixture of other enzymes, such as chymotrypsin, elastase, carboxypeptidase, kallikreins, and insulinase, whose effects on cell culture are not fully understood. These acetone extract preparations are subsequently mixed with lactose to adjust potencies. This trypsin has been successfully used in recent decades in the commercial-scale manufacture of tissue culture-based vaccines and other biological products. However, being a material of animal origin, it has the potential to introduce known or unknown adventitious agents, inherent to the source material, into the final product. Petition 870250085909, dated 09 / 23 / 2025, pp. 228 / 377 38 / 156

[0150] Up to 55 species of swine viruses from human hosts, from 17 different families, have been identified as potentially contaminating viruses. Even after extensive purification of animal-derived trypsin, however, contaminating activities exist in most preparations that can have undesirable consequences for both experimental research and the processing of pharmaceutical therapeutic proteins. These risks from various adventitious agents are typically mitigated by testing the swine trypsin for the absence of foreign agents; however, testing methods have their own limitations, and sometimes contaminating adventitious agents can escape detection.

[0151] In a more preferred embodiment, the enzyme includes recombinant trypsin.

[0152] Strict guidelines and regulations from regulatory bodies (e.g., the Food and Drug Administration, as well as other national and international regulatory bodies) have led to the need for pure trypsin of recombinant origin. Non-recombinant trypsin is associated with contamination. The rotavirus vaccine is prone to contamination with porcine circovirus DNA sequences originating from the porcine trypsin used during vaccine development.

[0153] The 2013 EMEA (European Medicines Agency) guidelines recommend the use of two different cell lines to test for adventitious agents that may be found in porcine trypsin. These guidelines also suggest the use of reagents free of animal components, such as recombinant trypsin, instead of animal-derived trypsin. Recombinant trypsin is a genetically modified protein, expressed in suitable microorganisms (E. coli, Pichia pastoris) and purified by high-performance liquid chromatography.

[0154] Recombinant trypsin is safer than porcine-derived trypsin and completely eliminates the risk of contamination by Petition 870250085909, dated 09 / 23 / 2025, pp. 229 / 377 39 / 156 reagents of animal origin.

[0155] In one embodiment, the trypsin used in the trypsinization step is a mixture of trypsin (0.25%, recombinant) and EDTA (0.005%). The trypsin solution is sterilized by 0.1 μ filtration and, similar to the growth medium, trypsin is also added to the TC flasks using an additional 0.1 μ filter (i.e., terminal filtration for each operation).

[0156] In one embodiment, recombinant trypsin with at least 95% purity and low endotoxin content facilitates the prevention of any adverse effects on the cell line. The recombinant trypsin used here has a molecular weight of 23 to 25 kilodaltons; Specific activity - 2500 USP units / mg.

[0157] In one embodiment, the addition of recombinant trypsin enzyme at optimum pH facilitates the effective breakdown of the protein, which dislodges the MRC-5 cell monolayer from the substrates (polystyrene, glass surfaces).

[0158] In one embodiment, the cells of the cell line are easily detached, with little or no clumping, compared to cells at acidic pH. When the cells are in the growth phase, in the optimum pH range of 7.4 to 7.8, effective trypsinization is achieved. The optimum pH range for the growth of MRC-5 cells is 7.4 to 7.8, which causes improved trypsinization. Trypsinization is significantly improved in cultures maintained at physiological pH throughout the incubation period. STEP c):

[0159] In one embodiment, the method of producing the clarified viral pool includes infecting the cell line with a virus to form an infected cell line. VIRUS

[0160] In another form, for the CVP production method, Petition 870250085909, dated 09 / 23 / 2025, pp. 230 / 377 40 / 156 The virus is selected from the group comprising single-stranded RNA viruses, positive-sense, negative-sense, enveloped and non-enveloped, belonging to the families Picornaviridae, Caliciviridae, Togaviridae, Matonaviridae, Flaviviridae, Coronaviridae, Retroviridae, Filoviridae, Bunyaviridae, Rhabdoviridae, Orthomyxoviridae, Arenaviridae, Paramyxoviridae, or combinations thereof. In another embodiment, the virus is selected from the group comprising single-stranded RNA viruses, positive-sense, enveloped and non-enveloped, belonging to the families Picornaviridae, Caliciviridae, Togaviridae, Matonaviridae, Flaviviridae, Coronaviridae, Retroviridae, or combinations thereof.

[0161] In another embodiment, the virus is selected from the group comprising single-stranded, negative-sense, enveloped RNA viruses belonging to the families Filoviridae, Bunyaviridae, Rhabdoviridae, Orthomyxoviridae, Arenaviridae, Paramyxoviridae, or a combination thereof.

[0162] In another embodiment, the virus is selected from the group comprising single-stranded, negative-sense, enveloped RNA viruses belonging to the family Paramyxoviridae.

[0163] In another embodiment, the virus is selected from the group comprising single-stranded, positive-sense, enveloped RNA viruses belonging to the family Matonaviridae.

[0164] In another embodiment, the virus is selected from, but not limited to, adeno-associated virus, Aichi virus, lyssavirus, Banna virus, Barmah forest virus, polyomavirus, Bunyamwera virus, herpesvirus, Chandipura virus, Chikungunya virus, cosavirus, Coxsackie virus, dengue virus, eastern chimpanzee foamy simian virus, eastern equine encephalitis virus, ebolavirus, echovirus, Epstein-Barr virus, hepatitis virus, Hantaan virus, Hendra virus, horsepox virus, adenovirus, astrovirus, coronavirus, cytomegalovirus, enterovirus, human immunodeficiency virus, human metapneumovirus Petition 870250085909, dated 09 / 23 / 2025, pp. 231 / 377 41 / 156 mano, human papillomavirus, parainfluenza, parvovirus, respiratory syncytial virus, human T-lymphotropic virus, human torovirus, influenza virus, Japanese encephalitis virus, arenavirus, Marburg virus, Langat virus, Lassa virus, lymphocytic choriomeningitis virus, Machupo virus, Mayaro virus, measles virus, Mokola virus, molluscum contagiosum virus, monkeypox virus, mumps virus, New York virus, Nipah virus, Norwalk virus, Orf virus, Oropouche virus, Pichinde virus, poliovirus, Punta Toro sandfly virus, Puumala virus, rabies virus, Rift Valley fever virus, Rosavirus, Ross River virus, rotavirus, rubella virus, Sagiyama virus, salivirus, Toscana virus, Seoul virus, tick-borne virus, vaccinia virus, varicella-zoster virus, Smallpox virus, vesicular stomatitis virus, vientovirus, West Nile virus, yellow fever virus, or Zika virus.

[0165] In another embodiment, the virus is an enveloped, single-stranded, negative-sense RNA virus belonging to the family Paramyxoviridae, selected from among the measles and mumps viruses.

[0166] In another embodiment, the virus is an enveloped, single-stranded, positive-sense RNA virus belonging to the Matonaviridae family, the rubella virus.

[0167] In another form, the viral vaccine comprises three viruses selected from among the measles, mumps, and rubella viruses.

[0168] In another form, the viral vaccine comprises three viruses selected from among measles morbillivirus, mumps orthorubillavirus and rubella rubivirus.

[0169] In another embodiment, the measles virus strain in the composition / formulation of the freeze-dried measles vaccine is selected from the group comprising the Edmonston strain, including the Schwartz, Edmonston-Zagreb, Moraten; CAM-70; TD 97; Leningrad-16; AIK-C and Shanghai 191 (Ji-191) strains.

[0170] In another form, the measles virus strain in the vaccine Petition 870250085909, dated 09 / 23 / 2025, pp. 232 / 377 42 / 156 measles vaccine, freeze-dried / lyophilized, is the Edmonston-Zagreb measles vaccine strain and was obtained from the Institute of Immunology Zagreb, Croatia.

[0171] In another embodiment, the mumps virus strain in the composition / formulation of a live attenuated, freeze-dried mumps vaccine is selected from the group comprising Jeryl-Lynn, RIT 4385, Leningrad-3, Leningrad-Zagreb (L-Zagreb), Urabe Am9, Hoshino strain, Torii strain and S79 Rubini.

[0172] In another embodiment, the mumps virus strain in the live attenuated, freeze-dried / freeze-dried mumps vaccine is the L-Zagreb mumps vaccine strain, obtained from the Institute of Immunology Zagreb, Croatia.

[0173] In another embodiment, the rubella virus strain in the composition / formulation of a live attenuated, lyophilized / freeze-dried rubella vaccine is selected from the group comprising the strains Wister RA 27 / 3, BRD-2, Matsuba, DCRB19, Takahashi, Matsuura and TO-336.

[0174] In another embodiment, the rubella virus strain in the freeze-dried live virus vaccine is the Wister RA 27 / 3 rubella vaccine strain, obtained from the Institute of Immunology Zagreb, Croatia.

[0175] The term “live” is used in its conventional meaning: a live virus is a virus that has not been inactivated, that is, a virus capable of replicating in permissive cells. A live attenuated vaccine virus is a virus that does not induce the disease caused by the corresponding wild-type virus in animals or humans and that is capable of inducing a specific immune response. Multiplicity of Infection (MOI)

[0176] In one embodiment, the cell line is infected with a virus to form an infected cell line. The viral infection is Petition 870250085909, dated 09 / 23 / 2025, pp. 233 / 377 43 / 156 achieved by adding viruses to the cell line (i.e., to the virus / cell ratio) at MOIs of 1:5 to 1:60. Low MOI or within a low MOI range is preferable to avoid the cytotoxicity observed at higher MOIs. Low MOI leads to greater viral infectivity. STAGE d):

[0177] In one embodiment, for the method of producing the clarified viral pool, the infected cell line is washed with viral medium and buffer. VIRAL MEDIA

[0178] In one embodiment, for the method of producing the clarified viral pool, the infected cell line is washed with viral medium and buffer. The viral medium acts as a washing medium. Viral media include basal media, enriched media, selective and indicator media, transport media, storage media, and carbon sources.

[0179] In one embodiment, the carbon source includes carbohydrates, glucose, glutamine, sucrose, dextrose, galactose, fructose, or combinations thereof.

[0180] In another embodiment, the viral medium (hereinafter referred to interchangeably as viral medium) is selected from Medium 199 (M199), Eagle Modified Medium by Dulbecco (DMEM), Minimum Essential Medium (MEM); Eagle Minimum Essential Medium (E-MEM), Hank's MEM (H-MEM), Iscove's Modified Dulbecco 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 Medium 8 (E8); StemFlex culture medium; Basal Medium of Airway Epithelial Cells; Alpha Modified Minimum Essential Medium (a-MEM); StemMacs iPS-Brew medium; TeSR-E8 medium, mTeSRI, mTeSR Plus; GMEM (Glasgow Minimum Essential Medium); Opti-MEM I; SmGM-2; Fibroblast growth medium / FGM; Growth medium without Petition 870250085909, dated 09 / 23 / 2025, pp. 234 / 377 44 / 156 StemPro-34 serum; mTESRI medium; ECGM-2 medium; Williams E medium; M254 medium; CnT07 medium; TNM-FH medium; basal medium for mammary epithelial cell growth (MEBM); medium for complete skeletal muscle and basal medium NeuroCult NS-A.

[0181] In another embodiment, the viral medium is added after infection with the virus for washing the infected cell lines in the manufacturing process.

[0182] In another embodiment, washing of cell lines infected by the viral medium is carried out after infection with the virus in the manufacturing process. BUMP

[0183] In one embodiment, for the CVP production method, a buffer is used.

[0184] The buffer includes NaHCO3, NaOH, NaCl, (4-(2-hydroxyethyl)-1-piperazinoethanesulfonic acid), i.e., HEPES, (piperazino-N,N'-bis(2-ethanesulfonic acid)), i.e., PIPES, or (2-(N-morpholino)ethanesulfonic acid), i.e., MES.

[0185] In a preferred embodiment, the buffer includes NaHCO3 (sodium bicarbonate).

[0186] In a preferred embodiment, the buffer, i.e., NaHCO3 (sodium bicarbonate), is used in a batch in the concentration range of 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.

[0187] In a more preferable embodiment, the buffer, i.e., NaHCO3 (sodium bicarbonate), is used in a batch in the concentration range of 1.0 to 1.9 g / L or 1.2 to 1.9 g / L.

[0188] In another method, the infected cell line is washed with viral medium and buffer.

[0189] In another embodiment, the cell line or the infected cell line in the exponential growth phase is cultured with the Petition 870250085909, dated 09 / 23 / 2025, pp. 235 / 377 45 / 156 readdition of the plug.

[0190] In another embodiment, the cell line or the infected cell line in the exponential growth phase is cultured with additional ventilation or aeration and the readdition of the buffer.

[0191] In another modality, readmission of the buffer is carried out by reinforcement with additional buffer, i.e., NaHCOs, or by providing an additional quantity of the buffer with the cellular medium or the viral medium, or both.

[0192] In one embodiment, the concentration of the enhanced buffer for the cellular medium or the viral medium, or both, during the readdition of the buffer, is in the range of 5% to 40%, or 5% to 35%, or 5% to 30%.

[0193] In one embodiment, the concentration of the enhanced buffer for the cellular medium or the viral medium, or both, during the readdition of the buffer, is in the range of 10% to 30%, or 15% to 30%.

[0194] In one embodiment, the concentration of the enhanced buffer for the cellular medium or the viral medium, or both, during the readdition 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.

[0195] In one embodiment, the total buffer concentration, including the enhanced buffer concentration, for the cellular medium or the viral medium, 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 batch.

[0196] In another embodiment, the total buffer concentration for the cellular medium or the viral medium, or both, including the enhanced 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 batch.

[0197] In a preferred embodiment, the total buffer concentration for the cellular medium or the viral medium, or both, including the concentration of the enhanced buffer, is in the range of 1.5 g / L to 2.0 g / L in Petition 870250085909, dated 09 / 23 / 2025, pp. 236 / 377 46 / 156 batch.

[0198] In one embodiment, the CVP preparation method includes providing the cell line cells with cell medium, buffer, and at least one supplement.

[0199] pH is a critical process parameter for cell growth. pH is controlled by adequate ventilation to allow gas exchange and increase the buffering capacity of the cell medium, adding an additional amount of buffer to the cell medium, which does not cause pH fluctuation, increases cell count and increases yield.

[0200] In one embodiment, the pH is maintained in the range of 4.0 to 8.0. In a preferred embodiment, the pH is maintained in the range of 5.0 to 8.0, or 6.0 to 8.0, or 6.5 to 8.0.

[0201] In a more preferred embodiment, the pH is maintained in the range of 6.7 to 7.9, or 6.9 to 7.9, or 7.0 to 7.9, or 7.1 to 7.9, or 7.1 to 7.8.

[0202] Therefore, pH is one of the critical parameters in the viral vaccine manufacturing process. Exposure of the virus to extreme pH levels inactivates it. It has been reported that the virus is stable at pH in the range of 6 to 8 and that progressive inactivation of the virus occurs on both sides. The physical stability of the virus is highly compromised in an acidic environment. Even small changes in pH affect the native confirmation of the virus.

[0203] pH regulation is particularly important immediately after cell seeding, when a new culture is establishing itself, and is generally achieved by one of two buffering systems: (i) a “natural” buffering system, where gaseous CO2 equilibrates with the 0 CO3 / HCO3 content of the culture medium and (ii) chemical buffering using a zwitterion called HEPES. Cultures that utilize natural bicarbonate / CO2 buffering systems Petition 870250085909, dated 09 / 23 / 2025, pp. 237 / 377 47 / 156 cells need to be maintained in an atmosphere of 5 to 10% CO2 in the air, usually provided in a CO2 incubator. Bicarbonate / CO2 is low cost, non-toxic and also provides other chemical benefits to the cells. HEPES has superior buffering capacity in the pH range of 7.2 to 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 gas atmosphere.

[0204] In one embodiment, cell growth is controlled by adding an additional amount of NaHCO3 to the cell medium, which does not cause pH fluctuation, increases cell count and increases yield.pH, as one of the critical parameters of the cell growth process, must be controlled by adequate ventilation to allow gas exchange and increase the buffering capacity of the cell medium. Adding an additional amount of NaHCO3 to the cell medium, which does not cause pH fluctuations, increases cell count and increases yield. STAGE e):

[0205] In one embodiment, the method of producing the clarified viral pool includes collecting the infected cell line in the medium to obtain a harvest.

[0206] In one embodiment, the method of producing the clarified viral pool includes collecting and, optionally, recollecting the infected cell line one or more times.

[0207] In a preferred embodiment, the harvest is carried out at least once, twice, three times, four times, five times, six times, seven times, eight times, nine times or ten times.

[0208] In a more preferable modality, harvesting is carried out at least twice. Harvesting is associated with obtaining a viral yield > 50%. The increase in yield is approximately 30 to 40% higher compared to the yield obtained by traditional methods. Petition 870250085909, dated 09 / 23 / 2025, pp. 238 / 377 48 / 156 onais.

[0209] In one embodiment, multiple sampling (one or more samples) leads to a viral yield > 50% by adding an optimum concentration of sodium bicarbonate in a medium with an optimum pH ranging from 6 to 8. OPTIONAL INACTIVATION: (FOR RABIES AND ROTAVIRUS)

[0210] In an alternative approach, collection is followed by virus inactivation.

[0211] In a preferred embodiment, the virus used for infection is a poliovirus or a rabies virus. The cell line used is a Vero cell line. Collection is followed by virus inactivation. Inactivation is performed by chemical methods (using chemicals such as β-propiolactone, formaldehyde) or physical methods (heat / thermal inactivation). STAGE f)

[0212] In one embodiment, the method of producing the clarified viral pool includes the addition of at least one stabilizer to the collection.

[0213] In one embodiment, stabilizers constitute an important part of the excipients. Consistent physicochemical properties of stabilizers help maintain the quality attributes of the final product, especially thermostability.

[0214] In one embodiment, at least one stabilizer includes at least one carbohydrate, at least one protein, one amino acid, or a combination thereof.

[0215] In one embodiment, the stabilizer is selected from the group consisting of at least one carbohydrate, at least one amino acid and at least one hydrolyzed protein.

[0216] According to the modalities, at least one carbohydrate as a stabilizer is selected from a group consisting of, but not limited to, natural carbohydrates, synthetic carbohydrates, po Petition 870250085909, dated 09 / 23 / 2025, pp. 239 / 377 49 / 156 thiols, glass transition facilitators, monosaccharides, disaccharides, trisaccharides, oligosaccharides and their corresponding sugar alcohols, polyhydroxylated compounds, such as carbohydrate derivatives and chemically modified carbohydrates, hydroxyethyl starch and sugar copolymers. Both natural and synthetic carbohydrates are suitable for use. Synthetic carbohydrates include, but are not limited to, those whose glycosidic linkage has been replaced by a thiol or carbon linkage. Both D and L forms of carbohydrates are used. The carbohydrate is either non-reducing or reducing. When a reducing carbohydrate is used, the addition of Maillard reaction inhibitors is preferred. Reducing carbohydrates suitable for use in the composition are those known in the art and include, but are not limited to, glucose, sucrose, maltose, lactose, fructose, galactose, mannose, maltulose and lactulose.Non-reducing carbohydrates include, but are not limited to, non-reducing glycosides of polyhydroxylated compounds selected from sugar alcohols and other linear-chain polyalcohols. Other useful carbohydrates include raffinose, stachyose, melezitose, dextran, celibose, mannobiose, and sugar alcohols. Sugar alcohol glycosides are preferably monoglycosides, in particular compounds obtained by the reduction of disaccharides such as lactose, maltose, lactulose, and maltulose. The glass-forming agent is selected from the group consisting of sucrose, mannitol, trehalose, mannose, raffinose, lactitol, lactobionic acid, glucose, maltulose, isomaltulose, maltose, lactose, sorbitol, dextrose, fructose, glycerol, D-sorbitol, and fucose, or a combination thereof.

[0217] In one embodiment, the carbohydrate is sorbitol. It is used to protect the infectivity of the virus during the lyophilization process. Typically, sorbitol is present in a concentration range of 1 to 20% (w / v), preferably in the range of 1 to 10% (w / v), and more preferably in the range of 3 to 6% (w / v). Also preferably, Petition 870250085909, dated 09 / 23 / 2025, pp. 240 / 377 50 / 156 D-sorbitol is present at a concentration of 5% (w / v).

[0218] In one embodiment, at least one amino acid as a stabilizer is selected from, but not limited to, the group of tricine, leucine, isoleucine, L-histidine, glycine, glutamine, L-arginine, L-arginine hydrochloride, lysine, L-alanine, tryptophan, phenylalanine, tyrosine, valine, cysteine, glycine, histidine, methionine, proline, serine, threonine, or a combination thereof.

[0219] In one embodiment, the amino acid used as a stabilizer is selected from the group consisting of tricine, L-arginine hydrochloride, L-histidine, and L-alanine as suitable amino acids, individually or in combination. The amino acid includes tricine at a concentration ranging from 0.1% to 2% by weight / volume (w / v), preferably from 0.1% to 1%, more preferably from 0.1% to 0.5%, most preferably equal to 0.3% (w / v). The amino acid includes L-histidine at a concentration ranging from 0.1% to 2% (w / v), preferably from 0.1% to 1%, more preferably from 0.1% to 0.5%, most preferably equal to 0.21% (w / v). The amino acid includes L-alanine in a concentration ranging from 0.01% to 1% by weight / volume, preferably between 0.05% and 0.5%, more preferably between 0.08% and 0.2%, most preferably equal to 0.1% (w / v).The amino acid includes L-arginine hydrochloride ranging from 0.1% to 10% weight / volume, preferably from 0.1% to 5%, more preferably from 0.1% to 3%, and even more preferably equal to 1.6% (w / v).

[0220] In one embodiment, at least one hydrolyzed protein as a stabilizer is selected from a group consisting of gelatin, lactalbumin hydrolysate, monosodium glutamate, collagen hydrolysate, keratin hydrolysate, peptides, casein hydrolysate and whey protein hydrolysate or protein such as serum albumin.

[0221] In another embodiment, the hydrolyzed protein is selected Petition 870250085909, dated 09 / 23 / 2025, pp. 241 / 377 51 / 156 of a group consisting of gelatin / gelatin in a concentration ranging from 0.1% to 10% by weight / volume, preferably between 0.1% and 5%, more preferably between 0.1% and 3%, most preferably equal to 2.5% (w / v) and lactalbumin hydrolysate in a concentration ranging from 0.1% to 2% by weight / volume (w / v), preferably between 0.1% and 1%, more preferably between 0.1% and 0.5%, most preferably equal to 0.35% (w / v), individually or in combination.

[0222] When used in this document, the term “gelatin” means a sterile, pyrogen-free protein preparation (e.g., fractions) produced by partial acid hydrolysis (type A gelatin) or partial alkaline hydrolysis (type B gelatin) of animal collagen, most commonly derived from bovine, porcine, and fish sources. Gelatin can be obtained in different molecular weight ranges. Hydrolyzed gelatin is an effective protein stabilizer due to its interaction with viral particles as well as its ability to inhibit surface adsorption. Hydrolyzed gelatin creates and maintains the desired structure / appearance of a lyophilized vaccine cake. Hydrolysis converts high molecular weight gelatin (> 100,000 Da) into low molecular weight gelatin (between 2,000 and 5,000 Da). Low molecular weight gelatin is less likely to stimulate gelatin-specific IgE than high molecular weight gelatin in vaccinated individuals.Recombinant sources of gelatin can also be used.

[0223] In one embodiment, the gelatin is of porcine origin, acquired in Europe (Germany and France).

[0224] In one embodiment, the method of preparing Stabilizer-1, i.e., gelatin-sorbitol stabilizer, involves the partial hydrolysis of gelatin, which can be achieved by autoclaving at 110 to 120 °C for 120 minutes. Preferably, the hydrolysis is carried out in a mixing vessel where it is possible to measure the actual temperature of the Petition 870250085909, dated 09 / 23 / 2025, pp. 242 / 377 52 / 156 product with a consistent degree of mixing and uniformity.

[0225] In one embodiment, Stabilizer-I, i.e., gelatin-sorbitol, is filtered using a depth filter, such as the Cuno filter or a 0.2 micron membrane filter. The 0.2 micron membrane filter offers advantages such as the possibility of testing integrity before and after application.

[0226] In one embodiment, Stabilizer-ll is composed of L-Histidine, L-Alanine, Tricine, L-Arginine hydrochloride and Lactalbumin hydrolysate.

[0227] In one embodiment, the virus collection and stabilizers (as mentioned in Table 1) are mixed in the specific ratio (v / v) of 80:20:10 (Collection: Gelatin-Sorbitol stabilizer (Stabilizer-I): Stabilizer-II) and the stabilized virus samples are collected and stored below -60 °C. Table 1: Components and their concentrations in the mass viral vaccine. Serial Number Component Final Concentration in Vaccine 1 Virus Harvest Base Gelatin-Sorbitol Stabilizer (Stabilizer-I) 2 Partially hydrolyzed gelatin 0.1 to 5% 3 D-sorbitol 1 to 10% Stabilizer-II 4 L-Histidine 0.1 to 1% 5 L-Alanine 0.01 to 1% 6 Tricin 0.1 to 1% 7 L-Arginine Hydrochloride 0.1 to 5% 8 Lactalbumin Hydrolysate 0.1 to 10% STEP g)

[0228] In one embodiment, the method of producing the clarified viral pool includes clarifying the collection to obtain a clarified viral pool. Petition 870250085909, dated 09 / 23 / 2025, page 243 / 377 53 / 156 each (CVP).

[0229] In one embodiment, the CVP is obtained by the clarified viral pool production method, in which the clarified viral pool is obtained after clarification of the collection. FILTERS

[0230] In one embodiment, the method of producing the clarified viral pool includes clarifying the sample using at least one filter.

[0231] In one embodiment, the sample is clarified / clarification is performed using at least one filter. In another embodiment, the sample is clarified / clarification is performed using a combination of two or more filters.

[0232] In another method, the sample is clarified / clarification is carried out using a combination of two or more filters arranged in series.

[0233] In another embodiment, the collection is clarified / clarification is carried out using a combination of two or more filters, where at least one filter has equal or different pore sizes.

[0234] In one embodiment, at least one filter has pore sizes in the range of 0.1 μ to 10.0 μ.

[0235] In another embodiment, at least one filter has pore sizes in the range of 0.1 μ to 9.5 μ, or in the range of 0.1 μ to 9.0 μ, or in the range of 0.1 μ to 8.5 μ, or in the range of 0.1 μ to 8.0 μ.

[0236] In another method, the collection is clarified / clarification is carried out using a combination of two or more filters, wherein the filters have equal pore sizes.

[0237] In another embodiment, the sample is clarified / clarification is carried out using at least one set of filters. The filter set includes a combination of two filters, wherein a first filter has a pore size in the range of 3.1 μ to 10.0 μ and a second filter has a pore size in the range of 0.1 μ to 3.0 μ. Petition 870250085909, dated 09 / 23 / 2025, pp. 244 / 377 54 / 156

[0238] In another method, the sample is clarified / clarification is carried out using at least two sets of filters.

[0239] The first set of filters includes a combination of two filters, wherein a first filter has a pore size in the range of 3.1 μ to 10.0 μ and a second filter has a pore size in the range of 0.1 μ to 3.0 μ. In a preferred embodiment, the first set of filters includes a first filter with a pore size in the range of 4 μ to 8 μ and a second filter with a pore size of 0.1 μ to 0.9 μ.

[0240] The second set of filters includes a combination of two filters, wherein a first filter has pores in the range of 3.1 μ to 10.0 μ and a second filter has pores in the range of 0.1 μ to 3.0 μ. In a preferred embodiment, the first set of filters includes a first filter with pores in the range of 4 μ to 8 μ and a second filter with pores in the range of 0.1 μ to 0.9 μ.

[0241] In one embodiment, the construction material of one or more filters used for clarification is selected from modified PVDF, cellulose acetate, and polypropylene. These filters are validated for the process.

[0242] In another approach, the clarification step is carried out using other techniques, such as ultracentrifugation, chromatography, precipitation and nanofiltration. EXPONENTIAL GROWTH PHASE

[0243] In one embodiment, the method of producing the clarified viral pool includes growing the cell line or the infected cell line in the exponential growth phase.

[0244] In one embodiment, as cells reach the exponential growth phase, they become more metabolically active and each cell produces the maximum amount of carbon dioxide. This excess carbon dioxide generated by the cells increases the level of dissolved carbon dioxide and causes a decrease Petition 870250085909, dated 09 / 23 / 2025, pp. 245 / 377 55 / 156 of the pH. The addition of sodium bicarbonate, along with ideal ventilation or aeration during the exponential growth phase, aids in adequate gas exchange, which facilitates the slow dissociation of sodium bicarbonate, i.e., the reduction of CO2, thus maintaining an ideal stable pH throughout the process. This does not alter the native form of the virus and prevents virus inactivation. VENTILATION / AERATION

[0245] In one embodiment, the method of producing the clarified viral pool includes culturing the cell line or infected cell line in the exponential growth phase with additional ventilation or aeration.

[0246] In one embodiment, excess CO2 can inhibit the respiratory reactions of cells and can act as an acid poison, crossing membranes and altering the pH of the intracellular compartment.

[0247] In one embodiment, ventilation / aeration is provided by culturing the cell line or infected cell line in containers with ventilated lids / vented covers / ventilation lids.

[0248] In one embodiment, ventilation / aeration is provided using vented MLTCF-10 with Corning vented caps (instead of the Milex filter used in the routine process). The use of vented MLTCF-10 with Corning vented caps results in the slow dissociation of sodium bicarbonate and maintains an alkaline pH.

[0249] In one embodiment, when ventilation / aeration is provided, the pH is maintained between 6.5 and 8.0 or 7.0 and 8.0 or 7.2 and 7.8. Here, cell growth must be controlled by adequate ventilation to allow gas exchange that does not cause pH fluctuation, increases cell count and increases yield.

[0250] In one embodiment, the use of ventilated MLTCF-10 with Petition 870250085909, dated 09 / 23 / 2025, pp. 246 / 377 Corning's 56 / 156 vented caps provide 40 to 120% higher cell throughput compared to the vented MLTCF-10 with Milex filters. RE-ADDITION OF BUMP

[0251] In one embodiment, the cell line or the infected cell line in the exponential growth phase is cultured with the readdition of the buffer.

[0252] In one embodiment, the cell line or the infected cell line in the exponential growth phase is cultured with additional ventilation or aeration and readdition of the buffer.

[0253] In a preferred embodiment, the buffer, that is, NaHCO3 (sodium bicarbonate), is used in a batch in the concentration range of 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.

[0254] In a more preferable embodiment, the buffer, that is, NaHCO3 (sodium bicarbonate), is used in a batch in the concentration range of 1.0 to 1.9 g / L, or 1.2 to 1.9 g / L.

[0255] In another method, the infected cell line is washed with viral medium and buffer.

[0256] In another modality, readmission of the buffer is carried out by reinforcement with additional buffer, i.e., NaHCOs, or by providing an additional quantity of the buffer with the cellular medium or the viral medium, or both.

[0257] In one embodiment, the concentration of the enhanced buffer for the cellular medium or the viral medium, or both, during buffer readmission, is in the range of 5% to 40%, or 5% to 35%, or 5% to 30%.

[0258] In one embodiment, the concentration of the enhanced buffer for the cellular medium or the viral medium, or both, during buffer readmission, is in the range of 10% to 30%, or 15% to 30%. Petition 870250085909, dated 09 / 23 / 2025, pp. 247 / 377 57 / 156

[0259] In one embodiment, the concentration of the enhanced buffer for the cellular medium or the viral medium, or both, during the readdition 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.

[0260] In one embodiment, the total buffer concentration, including the enhanced buffer concentration for the cellular medium or the viral medium, 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 batch.

[0261] In another embodiment, the total concentration of the buffer for the cellular medium or the viral medium, or both, including the concentration of the reinforced buffer, 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 batch.

[0262] In a preferred embodiment, the total buffer concentration for the cellular medium or the viral medium, or both, including the enhanced buffer concentration, is in the range of 1.5 g / L to 2.0 g / L in the batch. CLARIFIED VIRUS POOL (CVP)

[0263] In one embodiment, the cleared virus pool (CVP) is obtained by the method described herein.

[0264] In one embodiment, the CVP is obtained by the clarified virus pool production method, in which the clarified virus pool is obtained after clarification of the collection.

[0265] In one embodiment, the method of producing the clarified virus pool, wherein the clarified virus pool is obtained for a virus including measles morbillivirus, mumps orthorubulavirus, rubella rubivirus, adeno-associated virus, Aichi virus, lyssavirus, Banna virus, Barmah forest virus, polyomavirus, Bunyamwera virus, herpesvirus, Chandipura virus, Chikungunya virus, cosavirus, coxsackievirus, dengue virus, eastern chimpanzee ape foamy virus, eastern equine encephalitis virus, ebolavirus, Petition 870250085909, dated 09 / 23 / 2025, pp. 248 / 377 58 / 156 echovirus, Epstein-Barr virus, hepatitis virus, Hantaan virus, Hendra virus, horse pox virus, adenovirus, astrovirus, coronavirus, cytomegalovirus, enterovirus, human immunodeficiency virus, human metapneumovirus, human papillomavirus, parainfluenza, parvovirus, respiratory syncytial virus, human T-lymphotropic virus, human torovirus, influenza virus, Japanese encephalitis virus, arenavirus, Marburg virus, Langat virus, Lassa virus, lymphocytic choriomeningitis virus, Machupo virus, Mayaro virus, measles virus, Mokola virus, molluscum contagiosum virus, monkeypox virus, mumps virus, New York virus, Nipah virus, Norwalk virus, Orf virus, Oropouche virus, Pichinde virus, poliovirus, Punta Toro phlebovirus, Puumala virus, rabies virus, Valley fever virus Rift virus, rosavirus, Ross River virus, rotavirus, rubella virus, Sagiyama virus, salivirus, Tuscany virus, Seoul virus, tick-borne virus, vaccinia virus.Varicella-zoster virus, smallpox virus, vesicular stomatitis virus, Viento virus, West Nile virus, yellow fever virus, or Zika virus.

[0266] In one embodiment, CVP is obtained by the clarified virus pool production method, wherein the clarified virus pool is measles morbillivirus or measles virus.

[0267] In one embodiment, CVP is obtained by the clarified virus pool production method, wherein the clarified virus pool is mumps orthorubulavirus or mumps virus.

[0268] In one embodiment, CVP is obtained by the clarified virus pool production method, wherein the clarified virus pool is Rubivirus rubellae or rubella virus. MEASLES

[0269] In another embodiment, the method for producing a pool of clarified measles virus comprises: a. provide the cell line in the medium, the buffer and the su Petition 870250085909, dated 09 / 23 / 2025, pp. 249 / 377 59 / 156 Supplement; b. treat cells from the cell line with the enzyme; c. infect the cell line with measles morbillivirus to form a measles-infected cell line; d. Wash the measles-infected cell line with the viral medium and buffer; e. harvest the measles-infected cell line from the medium to obtain a sample; optionally, harvest the measles-infected cell line again; f. add the stabilizer to the harvest; and g. clarify the sample to obtain the clarified measles virus pool (CVP). Optionally, where the cell line or the infected cell line in the exponential growth phase is: - grown with additional ventilation or aeration, or - supplied with buffer or refills - both.

[0270] In one embodiment, the cell line used as a substrate for measles virus growth is the Human Lung Fibroblast (MRC-5) cell line.

[0271] In one embodiment, the enzyme is recombinant trypsin.

[0272] In one embodiment, the cell growth medium used for the growth of the measles virus is the Minimum Essential Medium.

[0273] In one embodiment, the cell growth medium used for measles virus growth is supported or supplemented.

[0274] In one embodiment, the supplements added to the cell growth medium used for measles virus growth are glutamine, fetal bovine serum, and sodium bicarbonate. Petition 870250085909, dated 09 / 23 / 2025, pp. 250 / 377 60 / 156

[0275] In one embodiment, the preparation of the cell factories comprises the reactivation of MRC-5 cells preserved in liquid nitrogen as a working cell bank. During the preparation of the working cell bank, freezing of the cells is performed at the population doubling level (PDL-19) in a plastic ampoule, i.e., a sterile disposable plastic ampoule, Nunc. Furthermore, the reactivated cells are cultured in a sterile disposable plastic tissue culture flask (Nunc) with cell growth medium, and the cells are incubated at 36 ± 1 °C. At each passage step, cell growth medium is added to the tissue culture flask through an additional 0.1 μm filter (i.e., terminal filtration for each operation). Antibiotics are not added at any stage of cell or viral growth.

[0276] In one embodiment, for the preparation of mass measles vaccine, the process of preparing the cell factories comprises the reactivation of MRC-5 cells preserved in liquid nitrogen as a working cell bank. During the preparation of the working cell bank, the cells are frozen at the population doubling level (PDL-19) in a plastic ampoule, i.e., a sterile disposable plastic ampoule, Nunc. Then, the reactivated cells are cultured in a sterile disposable plastic tissue culture flask (Nunc) with the cell growth medium and the cells are incubated at 36 ± 1 °C. At each step, the cell growth medium is added to the tissue culture flask through an additional 0.1 μm filter (i.e., terminal filtration for each operation). Antibiotics are not added at any stage of cell or viral growth.

[0277] In one embodiment, fetal bovine serum (FBS) added to the cell growth medium for the preparation of measles virus volume is in the range of 5% to 15%, preferably 8% to 13%, and Petition 870250085909, dated 09 / 23 / 2025, pp. 251 / 377 61 / 156 the FBS is irradiated with GAMMA radiation in the dose range of 20 to 50 kgy·

[0278] In one embodiment, fetal bovine serum (FBS) added to the cell growth medium for the preparation of measles virus mass is 10% to 12.5%.

[0279] In one embodiment, fetal bovine serum (FBS) added to the cell growth medium for the preparation of measles virus mass is 10%.

[0280] In one embodiment, fetal bovine serum (FBS) added to the cell growth medium for the preparation of measles virus mass is 12.5%.

[0281] In one embodiment, the viral medium used in the production process of a cleared measles virus pool is the Minimum Essential Medium with sodium bicarbonate and without glutamine, fetal bovine serum.

[0282] In one embodiment, sodium bicarbonate as a buffer in the viral medium is in the range of 1 to 2.5 g / L, 1.2 to 2.3 g / L, 1.4 to 2.1 g / L, 1.6 to 2.0 g / L and 1.8 to 2.0 g / L.

[0283] In one embodiment, the viral medium contains minimum essential medium (MEM), which differs from the cellular medium in that it does not contain fetal bovine serum (FBS). Therefore, it is used to replace the cellular medium during the washing of rolling flasks in order to eliminate fetal bovine serum, i.e., heterologous proteins. It also acts as a maintenance medium for infected MRC-5 cells in rolling flasks, in which the released virus is accumulated during the incubation of rolling flasks containing MRC-5 cells infected with the measles virus.

[0284] In one embodiment, FBS supplemented in the cell growth medium is treated with gamma irradiation.

[0285] In one embodiment, cell culture using the human lung fibroblast cell line (MRC-5), being a cell line Petition 870250085909, dated 09 / 23 / 2025, pp. 252 / 377 62 / 156 human diploid cell, requires maintenance of pH in the physiological range or slightly above (7.4 to 7.6), which is equally important, since at this pH, the cells are healthier and the binding sites (CD-46) for the virus would be in a more favorable form for infection. In addition, exposure of cells to low pH induces the "Early Contact Inhibition phenomenon," which affects cell growth and, consequently, viral production.

[0286] In one embodiment, trypsinization is performed after the formation of a confluent monolayer of cells using trypsin solution and transferred to the other tissue culture flask with the cell growth medium.

[0287] In one embodiment, the trypsin used in the trypsinization step is a mixture of trypsin (0.25%, recombinant) and EDTA (0.005%). The trypsin solution is sterilized by 0.1 μ filtration and, similar to the growth medium, trypsin is also added to the TC flasks through an additional 0.1 μ filter (i.e., terminal filtration for each operation).

[0288] In one embodiment, the recombinant trypsin used in the trypsinization step is derived from the bacterium E. coli (Source: Biogenomics) and the yeast Pichia pastoris (Source: Richcore).

[0289] The ideal pH range for the growth of MRC-5 cells was 7.4 to 7.8, which causes improved trypsinization. Trypsinization is significantly improved in cultures maintained at physiological pH throughout the incubation period. Cells are easily detached with little or no clumping compared to cells in acidic pH. This is because the cells are in the growth phase and the ideal pH for trypsin is in the range of 7.4 to 7.8.

[0290] In one embodiment, cells cultured in TC flasks are transferred to the next flask by trypsinization of the cells from one flask and divided into two flasks. In duplication Petition 870250085909, dated 09 / 23 / 2025, pp. 253 / 377 In the initial 63 / 156 duplication (up to PDL-26-27), confluence is achieved in about 4 days, while in subsequent duplications (PDL-28-32) it takes about 6 to 8 days. In PDL-26-27, 100% of the cells from the flasks are sampled for mycoplasma testing and karyotyping (cell identification). Cell passage continues until PDL-30 with a split ratio of 1:2. In PDL-30, cells from the TC flasks are seeded in a cell factory (Nunc, 6320 cm2) with 4 to 6 TC flasks per cell factory. This step provides 2 population duplications in a single step, compared to only one in the TC flasks. Thus, a considerable reduction in the handling of the TC flasks is achieved. Approximately 80 to 120 million cells are used to seed a CF, with about 1.8 to 2.0 L of MEM supplemented with FBS.After approximately 48 to 72 hours, or 2 to 3 days, a medium change is performed, and 72 hours after this change, the cells are used to seed rolling flasks or cell cube modules following trypsinization. The last duplication used for cell culture in vaccine production is PDL-33. This limit is based on the normal lifespan of 2 / 3 of MRC-5 cells, which is confirmed to be 50 population duplications.

[0291] In one embodiment, for the production of a clarified measles viral pool, i.e., for the preparation of mass measles vaccine, the use of MLTCF-10 and MLTCF-32 is carried out before viral infection.

[0292] In one embodiment, the cells are in suspension. Viral infection is performed by adding the working seed virus to each rolling flask at an MOI (i.e., virus / cell ratio) of 1:5 to 1:20. The virus-infected cells in the rolling flask are placed in a rolling apparatus (Bellco, USA / RBiolab, India) and incubated at 0.4 rpm at 36 ± 1 °C.

[0293] In one form, after approximately 40 to 44 hours of infection Petition 870250085909, dated 09 / 23 / 2025, pp. 254 / 377 64 / 156 In this step, the infected cell monolayers (in the rolling flasks) are washed twice using viral medium (FBS-free MEM) to remove traces of FBS and finally seeded with FBS-free MEM. The rolls are then incubated again at 0.7 rpm at 32 ± 1 °C.

[0294] During the production of the clarified measles viral pool, i.e., the routine production of measles virus after infection, infected cells are incubated at 36 ± 1 °C for 40 to 48 hours until washing. At this stage, it has been observed that the pH generally falls below 7.2. This indicates that the buffering capacity of the culture medium is insufficient to handle the acidity produced by the infected cells and cannot maintain the pH above the physiological level. Therefore, to increase the buffering capacity, the culture medium used in the preparation of the cell pool should be fortified with sodium bicarbonate (NaHCO3). Considering the seeding density per roll (40 to 60 million), MOI (i.e., virus / cell ratio) (1:8 to 1:12) and post-infection incubation period (40 to 48 hours), the CM used only for the preparation of the cell pool was added in the selected range of 1 to 2.5 g / L, 1.2 to 2.3 g / L, 1.4 to 2.1 g / L, 1.6 to 2.0 g / L and 1.8 to 2.0 g / L of NaHCO3.Increasing the concentration of NaHCO3 in the CM used for cell pool preparation increases the buffering capacity of the medium. The pH of the medium used before washing the infected culture flasks was evaluated and was not lower than 7.2. It was maintained between 7.4 and 7.6. The buffering capacity of the cell culture medium was increased by adding an additional amount of NaHCO3 to the cell culture medium, which does not cause pH fluctuation, increasing the cell count, i.e., increasing the cell yield or % yield.

[0295] Therefore, pH is one of the critical parameters in the viral vaccine manufacturing process. Exposure of the virus to extreme pH levels inactivates it. It has been reported that the measles virus is most stable at Petition 870250085909, dated 09 / 23 / 2025, pp. 255 / 377 65 / 156 pH 7.6, and progressive inactivation of the virus occurs on both sides. The physical stability of the measles virus is highly compromised in an acidic environment. Even small changes in pH affect the native confirmation of the virus. The infectivity of the measles virus also depends on the conformational stability of the viral proteins. The alkaline medium not only neutralizes the acid produced by infected cells but also increases the yield of the virus and its stability after its release into the medium. Therefore, maintaining the ideal pH throughout the process is inevitable to maintain its native confirmation. It has also been reported that the alkaline medium suppresses the production of defective particles.

[0296] In another embodiment, the cell line or the cell line infected with measles in the exponential growth phase is cultured with the readdition of the buffer.

[0297] In another embodiment, the cell line or cell line infected with measles in the exponential growth phase is cultured with additional ventilation or aeration and the readdition of buffer.

[0298] In one embodiment, the incubation of measles-infected cells is carried out at temperatures ranging from 30°C to 40°C, which affects virus growth. Virus growth depends on the temperature required for specific virus growth, resulting in a high viral yield.

[0299] In one embodiment, the antibiotic is not added at any stage of cell growth or virus growth. In another embodiment, the antibiotic is added at any stage of cell or viral growth.

[0300] In one embodiment, the exchange of the cellular medium is carried out during the preparation of the mass viral vaccine.

[0301] In another modality, the exchange of the cellular medium is not carried out. The exchange of the cellular medium is practically impossible, since au Petition 870250085909, dated 09 / 23 / 2025, pp. 256 / 377 66 / 156 increases the cost and number of operations, materials, unnecessary movement of people in cleanrooms and, ultimately, the chance of contamination. By avoiding the change of medium to MLTCF-10, the additional burden on cleanrooms can be reduced, which will contribute to the improvement of cGMP practices.

[0302] In one embodiment, the Nunc tissue culture flask, namely MLTCF-10, has two openings (0.2 μmillex filters, 50 mm diameter). MLTCF-10 flasks were used in the process, including the cultivation of MRC-5 in cell growth medium containing Hank's MEM supplemented with 10% FBS and a sodium bicarbonate concentration of 2.0 g / L. The pH was adjusted to 6.9 to 7.1 by CO2 gas purging before use. The seeded cell count was 100 to 160 million cells, and the temperature and incubation period were 36 ± 1 °C and 5 to 7 days, respectively. The cell yield obtained was 400 to 500 million cells. The usual pH variations observed during the incubation period were from 7.8 to 8 and 7.0 or less.

[0303] The Corning CS-10 tissue culture flask with vented caps has standard 33 mm screw caps with non-wettable membranes with 0.2 µm pores, sealed directly to the caps. Cultures were started from the same inocula (100 to 120 million cells). During the first 24 to 48 hours, when a lower pH (7.0 to 7.2) is required for fixation, the openings were hermetically sealed with sterile tape / aluminum foil to prevent gas exchange. After 48 hours, the openings were opened by removing the tape / metal foil to allow gas exchange. The pH remained in the range of 7.4 to 7.6 during the remainder of the incubation period.

[0304] It was observed that in MLTCF-10 with Milex filters (routine process), the pH fell below the physiological range, while in MLTCF-10. [Reference to document number 870250085909, dated 23 / 09 / 2025, page 257 / 377] In the 67 / 156 ventilated caps, the pH was maintained in the range of 7.6 to 7.78. The MLTCF-10 ventilated with Corning Vented caps produced 45 to 114% more cells than the MLTCF-10 ventilated with a Milex filter. Therefore, it was found that the vented caps provide adequate gas exchange due to the slow dissociation of sodium bicarbonate, maintaining a slightly alkaline pH, compared to the vented openings with Milex filters routinely used.

[0305] In cultures with Milex filters, where the pH fell below the non-physiological range (below 7.1), the lower cell growth (count) could not have been caused by simple nutrient depletion, since merely maintaining the pH in the range of 7.6 to 7.8 resulted in a higher cell count when vented lids were used for gas exchange. Therefore, physical removal of carbon dioxide is not necessary.

[0306] In one embodiment, pH adjustment by gas exchange and extra addition of NaHCO3 in the cellular medium is also applicable to other mammalian cells (e.g., Vero cells used for Rotavirus and Rabies vaccines).

[0307] The filling volume per vial of the final product (vaccine) in batches of 10 doses was reduced from 1.0 ml to 0.5 ml. The consumption of Blind Vaccine (diluent used to dilute the volume during mixing) was reduced by approximately 30%. These factors also contribute to increased productivity in the filling section.

[0308] In one embodiment, the rolls are then harvested based on the extent of infection to virus clearance. Generally, this occurs 3 times in the first 48 hours after washing and then once every 24 hours. About 5 to 10 times, preferably 6 to 7 times, the rolls are harvested, and collection is stopped when the number of cells in the roll vials drops to 20 to 30%.

[0309] In one mode, during each collection, the rolls are Petition 870250085909, dated 09 / 23 / 2025, pp. 258 / 377 68 / 156 bundles, each bundle containing 30 to 35 rolls. The virus from each bundle is collected in sterile disposable plastic bags, and stabilizers are added in the specified proportion.

[0310] In one embodiment, sterile disposable plastic bags are selected from ethylene vinyl acetate (EVA), ultra-low density polyethylene (ULDP) and other biologically acceptable (BOC) construction materials.

[0311] In one embodiment, the collected sample and the stabilizers are mixed in a specific ratio.

[0312] In one embodiment, the collected sample and the measles virus stabilizers (as mentioned in Table 2) are mixed in a specific ratio (v / v) of 80:20:10 (Collection: Gelatin-Sorbitol (Stabilizer-I): Stabilizer-II) and the stabilized virus samples are collected and stored below -60°C. Table 2: Components and their concentrations in the mass vaccination against the measles virus. Serial Number Component Final Concentration in Vaccine 1 Measles Virus Harvest Gelatin-Sorbitol Base (Stabilizer-I) 2 Partially hydrolyzed gelatin 0.1 to 5% 3 D-sorbitol 1 to 10% Stabilizer-II 4 L-Histidine 0.1 to 1% 5 L-Alanine 0.01 to 1% 6 Tricine 0.1 to 1% 7 L-Arginine Hydrochloride 0.1 to 5% 8 Lactalbumin Hydrolysate 0.1 to 10%

[0313] In another embodiment, the stabilized measles virus sample is clarified / clarification is performed using at least one set of filters. The filter set includes a combination of two filters, wherein a first filter has a pore size in the range of Petition 870250085909, dated 09 / 23 / 2025, pp. 259 / 377 69 / 156 3.1 μm to 10.0 μm and a second filter has a pore size in the range of 0.1 μm to 3.0 μm.

[0314] In one embodiment, the stabilized measles virus sample is then clarified using one of the following series or in combination - first filter with 5 μ to 7 μ filters and second filter with 0.1 μ to 2 μ filters in series, and first filter with 5 μ to 7 μ filters and second filter with 0.1 μ to 2 μ filters in series.

[0315] In one embodiment, the stabilized measles virus collection is then clarified using one of the following series or in combination - first filter with 6 μ filters and second filter with 0.45 μ filters in series, and first filter with 6 μ filters and second filter with 1.2 μ filters in series. The filtered samples are collected in one of the following collectors - 10 L sterile disposable medium bags (Stedim or Nalgene bags) as Clarified Virus Pools (CVPs).

[0316] In one embodiment, the stabilized measles virus collection is then clarified using a first filter with 6 μ filters and a second filter with 0.45 μ filters in series. The filtered samples are collected in one of the following collectors - 10 L sterile disposable media bags (Stedim or Nalgene bags) as Clarified Virus Pools (CVPs).

[0317] In one embodiment, uninfected control cultures are evaluated by means of tests that include: Microscopic examination; Test for the presence of mycoplasma; Karyology; Test for the presence of foreign hemodsorbent agents using - simian cells / human cells / human diploid cells.

[0318] In one embodiment, the cell line infected with the measles virus, i.e., MRC-5 cells, is evaluated by means of tests that include: Microscopic examination; Test for the presence of mycoplasma; Karyology; Test for the presence of foreign hemodsorbent agents using simian cells / human cells / diploid cells Petition 870250085909, dated 09 / 23 / 2025, pp. 260 / 377 70 / 156 human.

[0319] In one embodiment, the measles virus pool used as the harvested virus pool is evaluated by means of tests that include sterility testing, testing for the presence of mycoplasma, testing for viral content, and cell culture testing of the neutralized virus pool for adventitious agents using simian cells / human cells / human diploid cells.

[0320] In one embodiment, Clarified Virus Pools (CVPs) of measles virus are evaluated by means of tests that include sterility testing, identity testing, clarification testing, viral content testing and testing for the presence of mycoplasma and adventitious agents.

[0321] In one embodiment, the Clarified Virus Pools in sterile disposable plastic bags are stored in clean cold chambers and maintained at a temperature below -20 °C. These CVPs are subsequently used to prepare the final volume of the specific viral vaccine, immediately before filling and lyophilization.

[0322] In one embodiment, Clarified Virus Pools of measles virus in sterile disposable plastic bags are stored in clean cold chambers and maintained at a temperature below -20 °C. These CVPs are subsequently used to prepare the final volume for the specific viral vaccine, immediately prior to filling and lyophilization.

[0323] In one embodiment, the method for producing a pool of clarified measles virus, as described, mentions the use of an optimal concentration of sodium bicarbonate in the medium (1.0 to 2.5 g / L) and low MCI (i.e., virus / cell ratio) ranging from 1:5 to 1:20, which provides cell yields in the form of % yield or recovery of the virus obtained, selected from the groups above 10%, above 20%, above 30%, above 40%, above 50%. Petition 870250085909, dated 09 / 23 / 2025, pp. 261 / 377 71 / 156 and above 60%. As the yield increases, the doses per batch increase from 8 to 9 million doses to 13 to 16 million doses. It was found that the annual need for facilities for mass vaccine preparation was changed from two to one. The substantial amount of FBS used with the cell growth medium can be saved, i.e., 7.0 to 8.0 L of FBS per batch are saved. Annually, approximately 350.0 L of FBS are saved.

[0324] In one embodiment, during the production method of a clarified virus pool for measles that provides mass measles vaccine, the process for gathering the number of rolling vials during the collection step can be 50 rolling vials or more per batch. Typically, in a batch of 500 rolling bottles, 10 groups of 50 rolling bottles can be formed, or even 2 groups of 250 rolling bottles each.This provides advantages such as fewer aseptic connections and aliquots from a well-mixed and homogenized pool, as well as fewer samples. MUMPS

[0325] In one embodiment, the method for producing a pool of clarified mumps virus comprises: a. provide the cell line in the medium, the buffer, and the supplement; b. treat the cells of the cell line with the enzyme; c. infect the cell line with the mumps virus or to form a mumps-infected cell line; d. Wash the mumps-infected cell line with the medium and buffer; e. harvest the mumps-infected cell line from the medium to obtain the harvest; optionally, collect the mumps-infected cell line; f. add the stabilizer to the harvest; and Petition 870250085909, dated 09 / 23 / 2025, pp. 262 / 377 72 / 156 g. clarify the sample to obtain the clarified viral pool (CVP) of mumps. Optionally, where the cell line or the infected cell line in the exponential growth phase is: - grown with additional ventilation or aeration, or - Provided for readmission of the tampon or - both.

[0326] In one embodiment, the cell line used as a substrate for the growth of the mumps virus is the chicken embryo fibroblast (CEF) cell line.

[0327] In one embodiment, the enzyme is recombinant trypsin.

[0328] In one embodiment, the cell growth medium used for the growth of the mumps virus is the Minimum Essential Medium.

[0329] In one embodiment, the cell growth medium used for the growth of the mumps virus is supported or supplemented with the supplements.

[0330] In one embodiment, the supplements added to the cell growth medium used for the growth of the mumps virus are glutamine, fetal bovine serum, sodium bicarbonate, and neomycin sulfate.

[0331] In one embodiment, the preparation of the cell factories comprises the initial incubation of specified pathogen-free (SPF) chicken eggs at 37 ± 1 °C for 9 to 11 days. The incubated eggs are candling on the day of chicken embryo fibroblast (CEF) preparation. Live eggs are identified by the presence of healthy blood veins in the inner membrane of the eggs and by healthy embryonic movement. Non-fertile eggs are identified by the absence of veins, while dead eggs exhibit deficient veins and no embryonic movement / development. Live and healthy eggs are selected for the preparation of Petition 870250085909, dated 09 / 23 / 2025, pp. 263 / 377 73 / 156 CEF. These eggs are superficially disinfected with 70% isopropyl alcohol and taken to an aseptic area. The eggs are manually opened with the aid of forceps and the embryos are removed. The head, limbs, and viscera of the embryos are removed, and the embryos are chopped into small pieces. These fragments are initially collected in cooled (2 to 8°C) phosphate-buffered saline (PBS). Subsequently, the embryos are washed with a sufficient quantity of cooled PBS to remove RBCs.

[0332] In one embodiment, for the preparation of mass mumps vaccine, the cell factory preparation process comprises the initial incubation of specified pathogen-free (SPF) chicken eggs at 37 ± 1°C for 9 to 11 days. The incubated eggs are candling on the day of chicken embryo fibroblast (CEF) preparation. Live eggs are identified by the presence of healthy blood vessels in the inner membrane of the eggs and by healthy embryonic movement. Non-fertile eggs are identified by the absence of blood vessels, while dead eggs show deficient blood vessels and no embryonic movement / development. Live and healthy eggs are selected for CEF preparation. The surface of these eggs is disinfected with 70% isopropyl alcohol and they are taken to an aseptic area. The eggs are manually opened with the aid of forceps and the embryos are removed.The head, limbs, and viscera of the embryos are removed, and the embryos are chopped into small pieces. These pieces are initially collected in cooled phosphate-buffered saline (PBS) (2-8 °C). Subsequently, the embryos are washed with a sufficient quantity of cooled PBS to remove RBCs.

[0333] In one embodiment, fetal bovine serum (FBS) is added to the cell growth medium for the preparation of the virus mass. Petition 870250085909, dated 09 / 23 / 2025, pp. 264 / 377 74 / 156 of mumps is in the range of 5% to 15%, preferably 8% to 13%, and the FBS is irradiated with GAMMA radiation in the dose range of 20 to 50 kgy·

[0334] In one embodiment, fetal bovine serum (FBS) added to the cell growth medium for the preparation of mumps virus mass is 10% to 12.5%.

[0335] In one embodiment, fetal bovine serum (FBS) added to the cell growth medium for the preparation of mumps virus mass is 10%.

[0336] In one embodiment, fetal bovine serum (FBS) added to the cell growth medium for the preparation of the mumps virus volume is 12.5%.

[0337] In one embodiment, the viral medium used in the production process of a cleared mumps virus pool is Minimum Essential Medium with sodium bicarbonate and without glutamine, fetal bovine serum and neomycin sulfate.

[0338] In one embodiment, sodium bicarbonate in the viral medium is in the range of 1 to 2.5 g / L, 1.2 to 2.3 g / L, 1.4 to 2.1 g / L, 1.6 to 2.0 g / L and 1.8 to 2.0 g / L.

[0339] In one embodiment, the virus medium contains minimum essential medium (MEM), which differs from the cell medium by not containing fetal bovine serum (FBS) and neomycin sulfate. Therefore, it is used to replace the cell medium during the washing of cell factories / cell stacks in order to eliminate fetal bovine serum, i.e., heterologous proteins and neomycin sulfate. It also acts as a maintenance medium for infected CECs in cell factories / cell stacks, where the released virus accumulates during incubation.

[0340] In one embodiment, trypsinization is performed after washing the embryos with a sufficient quantity of cooled PBS to remove red blood cells. The embryos are suspended in a solution of Petition 870250085909, dated 09 / 23 / 2025, pp. 265 / 377 75 / 156 PBS and trypsin (both pre-warmed in a water bath at 36 °C) are placed in conical trypsinization flasks containing magnetic stir bars. Trypsinization is performed in an incubator at 36 °C on a magnetic stirrer for 30 to 40 minutes. After 24 hours of incubation, all culture vessels, i.e., production and control cultures, are replaced with fresh culture medium (MEM supplement with 5% to 15% FBS and neomycin). After incubation, chicken embryo fibroblast cells are routinely observed under a microscope to check growth progress.

[0341] In another embodiment, the cell line or the cell line infected with mumps in the exponential growth phase is cultured with the readdition of the buffer.

[0342] In another embodiment, the cell line or the cell line infected with mumps in the exponential growth phase is cultured with additional ventilation or aeration and the readdition of the buffer.

[0343] In one embodiment, after 2 to 3 days of incubation, viral infection of chicken embryo fibroblast cells is carried out by adding the working seed virus to the cells at a MOI (i.e., virus / cell ratio) of 1:20 to 1:60. The virus is adsorbed onto the monolayer for 1 hour at 33 ± 1 °C. After the adsorption period, the cell factories are again fed with MEM containing 10% FBS and Neomycin at a concentration of 50 ppm. After infection, incubation is continued at 33 ± 1 °C.

[0344] In one embodiment, after two days or 40 to 50 hours of infection, the medium from all cell factories is discarded and the infected cell monolayer (in the cell factories) is washed twice with FBS-free and neomycin-free MEM to remove traces of FBS and neomycin. After washing, the cell factories are re-fed with fresh FBS-free and neomycin-free MEM. The cell factories are then incubated again at 33 ± 1 °C. Petition 870250085909, dated 09 / 23 / 2025, pp. 266 / 377 76 / 156

[0345] In one embodiment, the virus accumulated in the cell factories is collected based on the extent of infection for virus removal. After collection, the cell factories are reseeded with fresh MEM without FBS. Incubation of the cell factories is continued at 33 ± 1 °C. A total of about 5 to 10 times, preferably 6 times, harvests are carried out with a frequency of one harvest per day.

[0346] In one embodiment, during each harvest, the virus from each group is collected in sterile disposable plastic bags and stabilizers are added in the specified proportion.

[0347] In one embodiment, sterile disposable plastic bags are selected from ethylene vinyl acetate (EVA), ultra-low density polyethylene (ULDP) and other biologically acceptable (BOC) construction materials.

[0348] In one embodiment, the mumps virus harvest and stabilizers (as mentioned in Table 3) are mixed in the specific ratio (v / v) of 80:20:10 (Harvest: Gelatin-Sorbitol (Stabilizer-I): Stabilizer-II) and the stabilized virus samples are collected and stored below -60°C. Table 3: Components and their concentrations in the mass mumps vaccine. Serial Number Component Final Concentration in Vaccine 1 Mumps Virus Harvest Gelatin-Sorbitol Base (Stabilizer-I) 2 Partially hydrolyzed gelatin 0.1 to 5% 3 D-sorbitol 1 to 10% Stabilizer-I 4 L-Histidine 0.1 to 1% 5 L-Alanine 0.01 to 1% 6 Tricine 0.1 to 1% 7 L-Arginine Hydrochloride 0.1 to 5% 8 Lactalbumin Hydrolysate 0.1 to 10% Petition 870250085909, dated 09 / 23 / 2025, pp. 267 / 377 77 / 156

[0349] In another embodiment, the stabilized mumps virus crop is clarified / clarification is carried out using at least one set of filters. The filter set includes a combination of two filters, wherein a first filter has a pore size in the range of 3.1 μ to 10.0 μ and a second filter has a pore size in the range of 0.1 μ to 3.0 μ.

[0350] In one embodiment, the stabilized mumps virus sample is then clarified using one of the following series or in combination - first filter with 5 μ to 7 μ filters and second filter with 0.1 μ to 2 μ filters in series, and first filter with 5 μ to 7 μ filters and second filter with 0.1 μ to 2 μ filters in series. In another embodiment, the stabilized mumps virus is then clarified using one of the following series or in combination - first filter with 6 μ filters and second filter with 0.45 μ filters in series, and first filter with 6 μ filters and second filter with 1.2 μ filters in series. The filtered samples are collected and stored in sterile disposable plastic bags as Clarified Virus Pools (CVPs).

[0351] In one embodiment, the stabilized mumps virus sample is then clarified using a first filter with 6 μ filters and a second filter with 1.2 μ filters in series. The filtered samples are collected and stored in sterile disposable plastic bags as Clarified Virus Pools (CVPs).

[0352] In one embodiment, uninfected control cultures for the production of the clarified viral pool of mumps virus are evaluated using tests that include: Microscopic examination; Test for the presence of mycoplasma; Karyology; Test for the presence of hemodsorbent foreign agents using simian cells / human cells / human diploid cells.

[0353] In one embodiment, the mumps viral pool or the harvested mumps viral pool is evaluated using tests that include: Test Petition 870250085909, dated 09 / 23 / 2025, pp. 268 / 377 78 / 156 sterility test, Mycoplasma presence test, Viral content test, Neutralized viral pool cell culture test for adventitious agents using simian cells / human cells / human diploid cells.

[0354] In one embodiment, Clarified Virus Pools (CVPs) of mumps virus are evaluated by means of tests that include sterility testing, identity testing, clarification testing, viral content testing, testing for the presence of mycoplasma and adventitious agents.

[0355] In one embodiment, Clarified Mumps Virus Pools in sterile disposable plastic bags are stored in clean cold chambers and maintained at a temperature below -20 °C. These CVPs are subsequently used to prepare the final volume for the specific viral vaccine, immediately prior to filling and lyophilization.

[0356] In one embodiment, in the method for producing a pool of clarified mumps virus, as described, which provides mass mumps vaccine, the SPF egg incubation process is carried out in an egg incubator with a capacity for 1,400 eggs or in an expanded version for up to 2,400 eggs, the latter being the advantage of improvised production capacity.

[0357] In one embodiment, the method for producing a pool of clarified mumps virus on a large scale, which provides mass mumps vaccine, is carried out using CF10 or CS10 and CF40 or CS40. All are sterile, disposable containers with gamma radiation for cell and virus growth, the latter providing almost four times the surface area for cultivation in a virtually equal space. This improvised scale-up method was established without impacting the productivity and quality attributes of the product through a validation process. Petition 870250085909, dated 09 / 23 / 2025, pp. 269 / 377 79 / 156 RUBELLA

[0358] In one embodiment, the method for producing a pool of clarified rubella virus comprises: a. provide the cell line in the medium, the buffer, and the supplement; b. treat the cells of the cell line with enzyme; c. infect the cell line with a Rubivirus rubellae virus to form a rubella-infected cell line; d. Wash the rubella-infected cell line with the medium and buffer; e. collect the rubella-infected cell line from the medium to obtain the sample; optionally, collect the rubella-infected cell line again; f. add the stabilizer to the harvest; and g. clarify the sample to obtain the clarified rubella virus pool (CVP). optionally, where the cell line or the rubella-infected cell line in the exponential growth phase is - grown with additional ventilation or aeration, or - supplied with the replacement of the plug or both.

[0359] In one embodiment, the cell line used as a substrate for rubella virus growth is the Human Lung Fibroblast (MRC-5) cell line.

[0360] In one embodiment, the enzyme is recombinant trypsin.

[0361] In one embodiment, the cell growth medium used for the growth of the rubella virus is Minimum Essential Medium.

[0362] In one embodiment, the cell growth medium used for rubella virus growth is supported or supplemented. Petition 870250085909, dated 09 / 23 / 2025, pp. 270 / 377 80 / 156

[0363] In one embodiment, the supplements added to the cell growth medium used for rubella virus growth are glutamine, fetal bovine serum, sodium bicarbonate, dextrose, and neomycin sulfate.

[0364] In one embodiment, the preparation of the cell factories comprises the reactivation of MRC-5 cells preserved in liquid nitrogen as a working cell bank. The cells are frozen at the population doubling level (PDL-19) in a plastic ampoule, i.e., a sterile disposable plastic ampoule, Nunc. Furthermore, the reactivated cells are cultured in a sterile disposable plastic tissue culture flask (Nunc) with cell growth medium, and the cells are incubated at 36 ± 1 °C. At each passage step, cell growth medium is added to the tissue culture flask through an additional 0.1 μm filter (i.e., terminal filtration for each operation). The cell growth medium is supplemented with antibiotic, which is subsequently removed during the washing step.

[0365] In one embodiment, for the preparation of mass rubella vaccine, the process of preparing the cell factories comprises the reactivation of MRC-5 cells preserved in liquid nitrogen as a working cell bank. During the preparation of the working cell bank, the cells are frozen at the population doubling level (PDL-19) in a plastic ampoule, i.e., a sterile disposable plastic ampoule, Nunc. Then, the reactivated cells are cultured in sterile disposable plastic tissue culture flasks (Nunc) with the cell growth medium and the cells are incubated at 36 ± 1 °C. At each step, the cell growth medium is added to the tissue culture flask through an additional 0.1 μm filter (i.e., terminal filtration for each operation). The growth medium Petition 870250085909, dated 09 / 23 / 2025, pp. 271 / 377 81 / 156 Cellular cement is supplemented with antibiotic, which is subsequently removed by washing.

[0366] In one embodiment, fetal bovine serum (FBS) added to the cell growth medium for the preparation of rubella virus mass is in the range of 5% to 15%, preferably 8% to 13%, and the FBS is irradiated with GAMMA radiation in the dose range of 20 to 50 kgy·

[0367] In one embodiment, fetal bovine serum (FBS) added to the cell growth medium for rubella virus mass preparation is in the range of 10% to 12.5%.

[0368] In one embodiment, fetal bovine serum (FBS) added to the cell growth medium for the preparation of rubella virus mass is 10%.

[0369] In one embodiment, fetal bovine serum (FBS) added to the cell growth medium for the preparation of rubella virus mass is 12.5%.

[0370] In one embodiment, the viral medium is the Minimum Essential Medium with sodium bicarbonate, dextrose, glutamine and, without fetal bovine serum, neomycin sulfate.

[0371] In one embodiment, sodium bicarbonate in the viral medium is in the range of 1 to 2.5 g / L, 1.2 to 2.3 g / L, 1.4 to 2.1 g / L, 1.6 to 2.0 g / L and 1.8 to 2.0 g / L.

[0372] In one embodiment, the viral medium contains minimum essential medium (MEM), which differs from the cell medium by not containing fetal bovine serum (FBS). Therefore, it is used to replace the cell culture medium during the washing of rolling flasks in order to eliminate fetal bovine serum, i.e., heterologous proteins. It also acts as a maintenance medium for infected MRC-5 cells in the cell cube system, in which the released virus is accumulated during the incubation of MRC-5 cells infected with the rubella virus. Petition 870250085909, dated 09 / 23 / 2025, pp. 272 / 377 82 / 156

[0373] In one embodiment, trypsinization is performed after the formation of a confluent monolayer of cells using trypsin solution and transferred to the other tissue culture flask with the cell growth medium.

[0374] In one embodiment, the trypsin used in the trypsinization step is a mixture of trypsin (0.25%, recombinant) and EDTA (0.005%). The trypsin solution is sterilized by 0.1 μ filtration and, similar to the growth medium, trypsin is also added to the TC flasks through an additional 0.1 μ filter (i.e., terminal filtration for each operation).

[0375] In one embodiment, the cell cube system is used for the mass preparation of rubella vaccine. This module consists of Cell Cube modules (sterile, disposable plastic, 85,000 cm2), an oxygenator bottle, and a Cell Cube system circulation pump. The medium in the Cell Cube system is continuously circulated (except after cell seeding) through the Cell Cube and oxygenator bottles via the circulation pump. The oxygenator bottle is supplied with compressed air, oxygen, and carbon dioxide. This helps maintain the desired pH and dissolved oxygen content of the medium at the defined value. Each Cell Cube module is a sterile, disposable 85,000 cm2 container containing 100 parallel polystyrene plates fused inside to facilitate and standardize the entry, distribution, and exit of the circulating medium. Four of these modules are integrated into each production batch.

[0376] In another embodiment, the cell line or the rubella-infected cell line in the exponential growth phase is cultured with the readdition of the buffer.

[0377] In another embodiment, the cell line or the rubella-infected cell line in the exponential growth phase is cultured with additional ventilation or aeration and the readdition of the buffer. Petition 870250085909, dated 09 / 23 / 2025, pp. 273 / 377 83 / 156

[0378] In one embodiment, after 7 to 8 days of incubation, the medium from the entire Cell Cube system is discarded / removed and viral infection is performed by adding the working seed virus to the cell monolayer at an MCI (i.e., virus / cell ratio) of 1:5 to 1:20. The virus is adsorbed onto the monolayer for 2 hours at 31 ± 1 °C. After the adsorption period, recirculation of the medium in the Cell Cube system is initiated and incubation proceeds at 31 ± 1 °C.

[0379] In one embodiment, after two days of incubation of virus-infected cells, the infected cell monolayer is washed with FBS-free MEM to remove traces of FBS and neomycin. After washing, the Cell Cube system is finally seeded with FBS-free MEM. The Cell Cube system is then incubated again at 31 ± 1 °C.

[0380] In one embodiment, the virus accumulated in the Cell Cube system is collected based on the extent of infection for virus removal and, after collection, the Cell Cube system is reseeded with MEM without FBS. Incubation of the Cell Cube system is continued at 31 ± 1 °C. A total of about 5 to 10 collections, preferably 8 to 9, are performed over a period of 8 to 10 days.

[0381] In one embodiment, during each collection, the virus from the entire Cell Cube system (including the oxygenator) is collected in pre-sterilized 50 L polypropylene bottles or in a 50 L culture medium bag, and stabilizers are added in the specified proportion.

[0382] In one embodiment, rubella virus collection and stabilizers (as mentioned in Table 4) are mixed in the specific ratio (v / v) of 80:20:10 (Collection: Gelatin-Sorbitol (Stabilizer-I): Stabilizer-II) and samples of the stabilized virus are collected and stored below -60°C. Table 4: Components and their concentrations in the vaccine in Petition 870250085909, dated 09 / 23 / 2025, page 274 / 377 84 / 156 against the rubella virus Serial No. Component Final Concentration in Vaccine 1 Rubella Virus Harvest Gelatin-Sorbitol Base (Stabilizer-I) 2 Partially hydrolyzed gelatin 0.1 to 5% 3 D-sorbitol 1 to 10% Stabilizer-II 4 L-Histidine 0.1 to 1% 5 L-Alanine 0.01 to 1% 6 Tricine 0.1 to 1% 7 L-Arginine Hydrochloride 0.1 to 5% 8 Lactalbumin Hydrolysate 0.1 to 10%

[0383] In another embodiment, the stabilized rubella virus harvest- The béola is clarified / clarification is carried out using at least one set of filters. The filter set includes a combination of two filters, wherein a first filter has a pore size in the range of 3.1 μ to 10.0 μ and a second filter has a pore size in the range of 0.1 μ to 3.0 μ.

[0384] In one embodiment, the stabilized rubella virus sample is then clarified using one of the following series or in combination - first filter with 5 μ to 7 μ filters and second filter with 0.1 μ to 2 μ filters in series and first filter with 5 μ to 7 μ filters and second filter with 0.1 μ to 2 μ filters in series. In one embodiment, clarification of the stabilized rubella virus sample is performed using a first 5 μ or 6 μ filter with a second 0.45 μ or 0.2 μ filter to obtain Clarified Virus Pools (CVPs). Final filtration with a 0.45 μ or 0.2 μ filter provides greater assurance of sterility.

[0385] In one embodiment, the clarification of the stabilized rubella virus collection is preferably performed with a first 6 μ filter and a second 0.45 μ filter to obtain Clarified Virus Pools (CVPs). The final filtration with a 0.45 μ filter provides greater assurance of sterility. Petition 870250085909, dated 09 / 23 / 2025, pages 275 / 377 85 / 156

[0386] In one embodiment, uninfected control cultures used for the production of the clarified viral pool of rubella virus are evaluated by means of tests that include: Microscopic examination; Test for the presence of mycoplasma; Karyology; Test for the presence of hemodsorbent foreign agents using simian cells / human cells / human diploid cells.

[0387] In one embodiment, the harvested rubella virus pool or rubella virus pool is evaluated by means of tests that include sterility testing, testing for the presence of mycoplasma, testing for viral content, and cell culture testing of the neutralized virus pool for adventitious agents using simian cells / human cells / human diploid cells.

[0388] In one embodiment, the Clarified Virus Pools (CVPs) of rubella virus are evaluated by means of tests that include sterility testing, identity testing, clarification testing, viral content testing and testing for the presence of mycoplasma and adventitious agents.

[0389] In one embodiment, Clarified Rubella Virus Pools in sterile disposable plastic bags are stored in clean cold chambers and maintained at a temperature below -20 °C. These CVPs are subsequently used to prepare the final volume for the specific viral vaccine, immediately prior to filling and lyophilization.

[0390] In one embodiment, the method for producing a pool of clarified rubella virus, which provides mass vaccination against the rubella virus, is carried out using rolling vials, cell factories or Cell Cube modules, all disposable single-use containers with gamma radiation. The Cell Cube offers advantages in terms of high surface area / volume ratio and therefore better yields in a nearly closed system, which is suitable for the principles Petition 870250085909, dated 09 / 23 / 2025, pp. 276 / 377 86 / 156 cipios of PAT. The Cell Cube is a parallel-plate bioreactor-based process that can be scaled up by integrating four Cell Cube modules of 85,000 cm2 each, to provide a total culture area of ​​340,000 cm2 in a small volume of up to 40 L, thus achieving viral concentration during the harvesting stage.

[0391] In one embodiment, the method for producing a pool of clarified rubella virus, the clarification of the stabilized rubella viral collection is performed using a 0.45 μ or 0.2 μ filter, providing better assurance of sterility. METHOD FOR OBTAINING LYOPHILIZED / FREEZE-DRIED MMR IMMUNOGENIC COMPOSITION:

[0392] In one embodiment, a method for obtaining a freeze-dried / lyophilized immunogenic composition for Measles, Mumps and Rubella (MMR) comprises one or more CVPs selected from a CVP for measles, a CVP for mumps, a CVP for rubella or a combination thereof, mixing the CVPs and then freeze-drying the combined CVP.

[0393] In one embodiment, a method for obtaining a freeze-dried / lyophilized immunogenic composition for Measles, Mumps and Rubella (MMR) comprises the mixing step, which is performed when a combination of CVPs is present in the immunogenic composition.

[0394] In one embodiment, a method for obtaining a freeze-dried / lyophilized immunogenic composition for Measles comprises mixing the CVP for measles with diluent and subjecting the mixed solution to the freeze-drying process.

[0395] In one embodiment, a method for obtaining a freeze-dried / lyophilized immunogenic composition against mumps comprises mixing mumps CVP with diluent and subjecting the mixed solution to the freeze-drying process. Petition 870250085909, dated 09 / 23 / 2025, pages 277 / 377 87 / 156

[0396] In one embodiment, a method for obtaining a freeze-dried / lyophilized immunogenic composition against rubella comprises mixing rubella CVP with diluent and subjecting the mixed solution to the lyophilization process.

[0397] In one embodiment, a method for obtaining a freeze-dried / lyophilized immunogenic composition against measles, mumps and rubella (MMR) comprises mixing one or more viruses in bulk, i.e., CVP, with diluent and subjecting the mixed solution to the freeze-drying process.

[0398] In one embodiment, a method for obtaining a freeze-dried / lyophilized immunogenic composition against measles, mumps and rubella (MMR) comprises mixing a measles CVP, a mumps CVP and a rubella CVP with diluent and subjecting the mixture to the freeze-drying process.

[0399] In one embodiment, the method for obtaining a freeze-dried / lyophilized immunogenic composition against measles, mumps and rubella (MMR) comprises mixing a measles CVP, a mumps CVP and a rubella CVP with diluent and, optionally, subjecting the mixture to the lyophilization process.

[0400] In one embodiment, the method for obtaining a freeze-dried / lyophilized immunogenic composition against measles, mumps and rubella (MMR) comprises the following steps: a) the CVPs of the measles, mumps and rubella viruses are kept for thawing at 30 to 35 °C in an incubator, and the mixing of the multiple components present in the CVPs and the vaccine is carried out blindly, obtaining a homogeneous solution as a volume of the final product; b) the homogeneous mass of the final product obtained is clarified through a 0.45 µm filter; Petition 870250085909, dated 09 / 23 / 2025, pp. 278 / 377 88 / 156 c) The homogeneous mass of the final product is aseptically packaged in sterilized bottles and transferred to trays. d) The trays are transferred to the freeze dryer for the freeze-drying / freeze-drying process. e) Freeze-drying of the homogeneous mass of the final product contained in the bottles obtained in step d), comprising the steps of freezing, sublimation and secondary drying.

[0401] In one embodiment, the method for obtaining a freeze-dried / lyophilized immunogenic composition against Measles, Mumps and Rubella (MMR) comprises the following steps: a) Thawing of measles, mumps, and rubella virus samples at 30 to 35 °C; b) mixing the thawed CVPs of measles, mumps, and rubella viruses with the vaccine in a blind mixing process to obtain a homogeneous solution; c) clarification of the homogeneous solution through a 0.45 pm filter to obtain a homogeneous mass; d) Aseptically fill the homogeneous mass into sterilized vials, followed by transferring the vials to a freeze dryer / freeze-dryer. (e) freeze-drying / lyophilization of the vials containing the homogeneous mass, where freeze-drying / lyophilization comprises freezing, primary drying / sublimation, and secondary drying.

[0402] In one embodiment, the CVPs of measles, mumps, and rubella viruses are removed from storage at -20°C and kept in an incubator at 30 to 35°C for thawing, in order to mix the multiple components present in the CVPs and the blind vaccine. The blind vaccine consists of the diluent used for dilution of the volume. Petition 870250085909, dated 09 / 23 / 2025, pp. 279 / 377 89 / 156 during mixing. The CVPs and the blind vaccine are mixed to prepare a homogeneous mass of the final product, which is clarified through a 0.45μ filter. The mixing of the above components is carried out in a stainless steel tank equipped with a magnetic stirrer and a jacket in which water circulates at 33°C. The mixed solution is aseptically filled into sterilized vials in a filling machine, a sterilized freeze-drying stopper is partially inserted into the neck of the vial, and the vials are transferred to trays. These trays are transferred to the freeze-dryer for the freeze-drying / freeze-drying process.

[0403] In one embodiment, the freeze-drying / lyophilization method comprises: a) pre-freezing shelf, loading the trays containing the vials of the immunogenic composition onto the shelf; b) freezing; c) primary drying / sublimation d) secondary drying.

[0404] In one embodiment, the method for obtaining a freeze-drying / lyophilization step comprises: (e) Pre-freezing shelf includes freezing from -35°C to -60°C. f) freezing stage comprising freezing at -50°C to -60°C for 450 to 650 minutes; g) primary drying / sublimation stage comprising acceleration from +0.5°C / minute to 1.0°C / minute at a temperature of 30°C for 1200 to 1700 minutes at 80 to 120 bar. and h) Secondary drying stage, comprising raising the temperature by +0.5°C / minute to 1.0°C / minute to reach a shelf temperature of 20 to 30°C, maintaining it for 300 to 500 minutes at 20°C. Petition 870250085909, dated 09 / 23 / 2025, pp. 280 / 377 90 / 156 at 35 pbar.

[0405] In one embodiment, the freeze-drying / lyophilization process comprises the steps of freezing, sublimation, and secondary drying. Trays of vials are loaded onto a pre-frozen shelf at -40°C. The shelf is heated to 50°C and maintained until the product reaches -40°C. The condenser is cooled below -60°C. After a specific pre-freezing period, the sublimation process is initiated by applying vacuum and starting a controlled heating cycle. After sublimation, the product undergoes secondary drying at 23°C and vacuum levels of 25 to 30 pbar for 7 hours. At the end of secondary drying, the product is capped at approximately 25 microbar absolute vacuum. After sealing, the vacuum is broken with 0.2 micron filtered air, and the product is discharged for sealing with an aluminum cap.The freeze-drying cycle consistently produced the lowest freeze-drying loss, constant thermostability value, residual moisture, and consistent solubility characteristics.

[0406] In one embodiment, the freeze-dried / lyophilized MMR immunogenic composition comprises at least one virus; a stabilizer comprising at least one carbohydrate, at least one amino acid and at least one hydrolyzed protein.

[0407] In one embodiment, the freeze-dried / lyophilized MMR immunogenic composition comprises: live attenuated measles virus present in a dose of not less than 1000 CCIDso per dose (0.5 mL), live attenuated mumps virus present in a dose of not less than 5000 CCIDso per dose (0.5 mL) and live attenuated rubella virus present in a dose of not less than 1000 CCIDso per dose (0.5 mL).

[0408] In one embodiment, the freeze-dried / freeze-dried MMR immunogenic composition comprises at least one carbohydrate Petition 870250085909, dated 09 / 23 / 2025, pp. 281 / 377 91 / 156 selected carbohydrate from a group consisting of natural carbohydrate, synthetic carbohydrate, monosaccharides, disaccharides, trisaccharides, oligosaccharides, reducing sugar, non-reducing sugar, sugar alcohols, polyol, polyhydroxylated compounds, chemically modified carbohydrates and glass transition facilitators, which include sucrose, mannitol, trehalose, mannose, raffinose, lactitol, lactobionic acid, glucose, maltulose, isomaltulose, maltose, lactose, sorbitol, dextrose, fructose, glycerol, sorbitol and fucose, and a combination thereof; at least one carbohydrate in the concentration range of 1 to 20% (w / v); at least one of the carbohydrates is present in a concentration of 1 to 20% (w / v), 1 to 10% (w / v), preferably 3 to 6% (w / v).

[0409] In one embodiment, the freeze-dried / lyophilized MMR immunogenic composition comprises at least one amino acid selected from a group consisting of tricine, leucine, isoleucine, 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; at least one amino acid in the concentration range of 0.01 to 10% (w / v); At least one of the amino acids 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).

[0410] In one embodiment, the freeze-dried / lyophilized MMR immunogenic composition comprises at least one hydrolyzed protein obtained by chemical, enzymatic or thermal hydrolysis of plant or animal-derived proteins.

[0411] In one embodiment, the freeze-dried / lyophilized MMR immunogenic composition comprises at least one protein Petition 870250085909, dated 09 / 23 / 2025, pp. 282 / 377 92 / 156 in the selected hydrolysate from a group consisting of gelatin, lactalbumin hydrolysate, monosodium glutamate, collagen hydrolysate, keratin hydrolysate, peptides, casein hydrolysate and whey protein hydrolysate; at least one hydrolyzed protein in the concentration range of 0.1 to 10% (w / v); at least one of the hydrolyzed proteins selected from a group consisting of gelatin present in a concentration of 0.1% to 5% (w / v) and lactalbumin hydrolysate present in a concentration of 0.1% to 2% (w / v).

[0412] In one embodiment, the freeze-dried / lyophilized MMR immunogenic composition comprises measles, mumps, and rubella antigen with the excipients mentioned in Table 5; Table 5: Components and their concentration ranges in the immunogenic composition of live attenuated lyophilized / freeze-dried MMR (measles, mumps, and rubella vaccine (MMR)) Component Quantity Measles virus Not less than 1000 CCIDso / dose Mumps virus Not less than 5000 CCIDso / dose Rubella virus Not less than 1000 CCIDso / dose Gelatin (partially hydrolyzed) 0.1 to 5% D-sorbitol 1 to 10% L-histidine 0.1 to 1% L-alanine 0.01 to 1% Tricine 0.1 to 1% Arginine 0.1 to 5% Lactalbumin hydrolysate 0.1 to 10% Minimum Essential Medium (MEM) Base

[0413] In one embodiment, the freeze-dried / lyophilized MMR immunogenic composition comprises an adjuvant selected from a group consisting of aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, and aluminum potassium sulfate or Petition 870250085909, dated 09 / 23 / 2025, pp. 283 / 377 93 / 156 a mixture of the same.

[0414] In one embodiment, the freeze-dried / lyophilized MMR immunogenic composition comprises an immunostimulatory component selected from a group consisting of an oil-in-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, complete Freund's adjuvant, incomplete Freund's adjuvant, CRL-8300 adjuvant, muramyl dipeptide, TLR-4 agonists, flagellin, flagellins derived from Gram-negative bacteria, TLR-5 agonists, flagellin fragments capable of binding to TLR-5 receptors, QS-21, ISCOMS, chitosan, a combination of saponins with sterols and lipids.

[0415] In one embodiment, the freeze-dried / freeze-dried MMR immunogenic composition comprises a pharmaceutically acceptable additive selected from a group consisting of a carrier, excipient, binder, isotonic agent, emulsifier and humectant.

[0416] In one embodiment, the freeze-dried / freeze-dried MMR immunogenic composition comprises an excipient selected from a group consisting of salt including NaCl, KCl, KH2PO4, Na2HPO4.2H2O, CaCb and MgCb; non-ionic surfactant, including polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, oxtoxinol 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, and cyclodextrin. Petition 870250085909, dated 09 / 23 / 2025, pp. 284 / 377 94 / 156

[0417] In one embodiment, the lyophilized / freeze-dried MMR immunogenic composition is reconstituted with an aqueous solution selected from a group consisting of saline, buffer and WFI (water for injection).

[0418] In one embodiment, the buffering agent in the freeze-dried / freeze-dried MMR immunogenic composition is selected from a group consisting of HEPES, citrate-phosphate, carbonate, phosphate, citrate, lactate, gluconate, borate, histidine buffer, succinate buffer, and tartrate buffering agents, as well as more complex organic buffering agents, including a phosphate buffering agent containing sodium phosphate and / or potassium phosphate in a ratio selected to achieve the desired pH. As an amino acid, histidine acts as a free radical scavenger and also stabilizes viral proteins through non-covalent histidine-protein interactions in the solid state.

[0419] In one embodiment, the freeze-dried / lyophilized MMR immunogenic composition comprises a buffer selected from a group consisting of sodium chloride, acetate, carbonate, citrate, lactate, gluconate, tartrate, saline phosphate buffer, borate, histidine buffer, succinate buffer, HEPES, TRIS, and citrate-phosphate.

[0420] In one embodiment, the dose of the lyophilized / freeze-dried MMR immunogenic composition is 0.5 mL.

[0421] In one embodiment, the final pH of the reconstituted freeze-dried / lyophilized MMR immunogenic composition is in the pH range of 6.5 to 7.5.

[0422] In one embodiment, the freeze-dried / freeze-dried MMR immunogenic composition is in the form of a single-dose composition and is preservative-free.

[0423] In another embodiment, the freeze-dried / freeze-dried MMR immunogenic composition is in the form of a composition Petition 870250085909, dated 09 / 23 / 2025, pages 285 / 377 95 / 156 multidose, and the multidose composition further comprises a preservative selected from the group comprising 2-phenoxyethanol, benzethonium chloride (femerol), phenol, m-cresol, thimerosal, formaldehyde, paraben esters (e.g., methyl-, ethyl-, propyl- or butylparaben), benzalkonium chloride, benzyl alcohol, chlorobutanol, pchloro-m-cresol or benzyl alcohol, or a combination thereof. The freeze-dried / lyophilized viral vaccine composition includes material for a single immunization or includes material for multiple immunizations (i.e., a “multidose” kit). The inclusion of a preservative is preferred in multidose arrangements. As an alternative (or in addition) to the inclusion of a preservative in multidose compositions, the compositions are contained in a container with an aseptic adapter for material removal.

[0424] In one embodiment, the freeze-dried / freeze-dried MMR immunogenic composition comprises: a) live attenuated measles virus present in a dose of not less than 1000 CCIDso per dose, live attenuated mumps virus present in a dose of not less than 5000 CCIDso per dose and live attenuated rubella virus present in a dose of not less than 1000 CCIDso per dose; b) stabilizer comprising: carbohydrate consisting of sorbitol present in a concentration of 1 to 10% (w / v); amino acid consisting of portricin present in a concentration of 0.1% to 2% (w / v), L-histidine present in a concentration of 0.1% to 2% (w / v), L-alanine present in a concentration of 0.01% to 1% (w / v) and L-arginine hydrochloride present in a concentration of 0.1% to 5% (w / v); and hydrolyzed protein consisting of gelatin present in a concentration of 0.1% to 5% (w / v) and lactalbumin hydrolysate present in a concentration of 0.1% to 2% (w / v).

[0425] In one embodiment, the lyophilized MMR immunogenic composition Petition 870250085909, dated 09 / 23 / 2025, pp. 286 / 377 96 / 156 freeze-dried / freeze-dried comprises: a) live attenuated measles virus present in a dose of not less than 1000 CCID50 per dose, live attenuated mumps virus present in a dose of not less than 5000 CCID50 per dose and live attenuated rubella virus present in 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).

[0426] In one embodiment, the final volume of the lyophilized / freeze-dried live attenuated MMR immunogenic composition is evaluated by means of tests comprising sterility test, test for presence of mycoplasma, test for viral content and test for residual animal serum.

[0427] In one embodiment, the final product of the lyophilized / freeze-dried live attenuated MMR immunogenic composition is evaluated by means of tests that include sterility test, test for presence of mycoplasma, identity test, general safety test, moisture content, test for viral content (potency) and thermostability (accelerated temperature test).

[0428] In one embodiment, storage and utilization methods for various intermediate media and reagents are improved, increasing the reliance on single-use disposable containers and Petition 870250085909, dated 09 / 23 / 2025, pages 287 / 377 97 / 156 sterilized by gamma radiation, with advantages such as reduced workload in validated cleaning procedures and autoclave sterilization. Examples of this are the use of medium bags with capacities of 20 L and 50 L for the collection and storage of cellular and viral media to be used in the preparation of medicinal substances and blind vaccines. Thus, the number of glass vials and stainless steel tanks used in medium preparation was significantly reduced and replaced by USP Class VI plastic bags, without affecting the quality attributes of the product.

[0429] In one embodiment, the live attenuated, lyophilized / freeze-dried MMR immunogenic composition comprising measles, mumps and rubella antigens is considered stable at -20°C, 2°C to 8°C, 25°C and 37°C for different time intervals / periods.

[0430] In one embodiment, the live attenuated, lyophilized / freeze-dried MMR immunogenic composition is administered to a human individual parenterally (subcutaneous, intradermal, intramuscular, intraperitoneal, intravenous, injectable, pulmonary, suppository, needle-free injection, transcutaneous administration) or sustained release from implants, or administration by eye drops, or administration via mucosal routes (oral, intranasal, pulmonary, rectal or vaginal), or administration via buccal, perioral, intragastric, perilingual, alveolar, gingival, olfactory or respiratory routes, or via interthecal, intralymphatic routes, by bladder instillation, by scarification or by any other routes of immunization.

[0431] In one embodiment, the final product of the freeze-dried / freeze-dried live attenuated immunogenic composition is maintained in a vaccine kit, where the kit comprises: a) a first container containing a freeze-dried / lyophilized viral vaccine composition / formulation comprising: at least one virus (antigen) selected from a Petition 870250085909, dated 09 / 23 / 2025, pp. 288 / 377 98 / 156 group consisting of measles virus, mumps virus and rubella virus; other excipients; and b) a second container containing an aqueous solution selected from saline solution or water for injection (WFI) for reconstitution of the freeze-dried viral vaccine composition.

[0432] In one embodiment, the final product of the freeze-dried / freeze-dried live attenuated immunogenic composition is maintained in a vaccine kit, where the kit comprises: a) a first container containing a freeze-dried / lyophilized viral vaccine composition / formulation comprising: at least two viruses (antigens) selected from a group consisting of measles virus, mumps virus and rubella virus; other excipients; and b) a second container containing an aqueous solution selected from saline solution or water for injection (WFI) for reconstitution of the freeze-dried (lyophilized) virus vaccine composition.

[0433] In one embodiment, the final product of the freeze-dried / freeze-dried live attenuated immunogenic composition is maintained in a vaccine kit, wherein the kit comprises: a) a first container containing a freeze-dried / lyophilized virus vaccine composition / formulation comprising: at least three viruses (antigens) selected from a group consisting of measles virus, mumps virus and rubella virus; other excipients; and b) a second container containing an aqueous solution selected from saline solution or water for injection (WFI) for reconstitution of the freeze-dried (lyophilized) virus vaccine composition. Petition 870250085909, dated 09 / 23 / 2025, pp. 289 / 377 99 / 156

[0434] In one embodiment, the final product of the live attenuated, lyophilized / freeze-dried immunogenic composition against Measles (M) is maintained in a vaccine kit, wherein the kit comprises: a) a first container containing a freeze-dried / lyophilized viral vaccine composition / formulation comprising: measles virus (antigen); other excipients; and b) a second container containing an aqueous solution selected from saline solution or water for injection (WFI) for reconstitution of the freeze-dried viral vaccine composition.

[0435] In one embodiment, the final product of the live attenuated, lyophilized / freeze-dried immunogenic composition against Measles and Rubella (MR) is maintained in a vaccine kit, wherein the kit comprises: a) a first container containing a freeze-dried / lyophilized viral vaccine composition / formulation comprising: measles and rubella viruses (antigens); other excipients; and b) a second container containing an aqueous solution selected from either saline solution or water for injection (WFI) for reconstitution of the freeze-dried (lyophilized) virus vaccine composition.

[0436] In one embodiment, the final product of the lyophilized / freeze-dried live attenuated immunogenic composition against Measles, Mumps and Rubella (MMR) is maintained in a vaccine kit, wherein the kit comprises: a) a first container containing a freeze-dried / lyophilized virus vaccine composition / formulation comprising: measles, mumps and rubella viruses (antigens); other excipients; and Petition 870250085909, dated 09 / 23 / 2025, pp. 290 / 377 100 / 156 b) a second container containing an aqueous solution selected from either saline solution or water for injection (WFI) for reconstitution of the freeze-dried (lyophilized) virus vaccine composition.

[0437] In one embodiment, a kit comprising the freeze-dried / freeze-dried MMR immunogenic composition comprises: (a) a first container containing a freeze-dried viral vaccine composition, comprising said composition: live attenuated measles virus present in a dose of not less than 1000 CCIDso per dose, live attenuated mumps virus present in a dose of not less than 5000 CCIDso per dose and live attenuated rubella virus present in a dose of not less than 1000 CCIDso per dose; carbohydrate consisting of sorbitol present in a concentration of 1 to 10% (w / v); amino acid consisting of portricin present in a concentration of 0.1% to 2% (w / v), L-histidine present in a concentration of 0.1% to 2% (w / v), L-alanine present in a concentration of 0.01% to 1% (w / v) and L-arginine hydrochloride present in a concentration of 0.1% to 5% (w / v); and hydrolyzed protein, consisting of gelatin present in a concentration of 0.1% to 5% (w / v) and hydrolyzed lactalbumin present in a concentration of 0.1% to 2% (w / v); and b) a second container containing an aqueous solution selected from saline solution or water for injection (WFI) for reconstitution of the lyophilized (freeze-dried) vaccine composition.

[0438] In another embodiment, a kit comprising the freeze-dried / freeze-dried MMR immunogenic composition comprises: a) a first container containing a lyophilized (freeze-dried) viral vaccine composition, wherein said composition comprises: live attenuated measles virus present in Petition 870250085909, dated 09 / 23 / 2025, pp. 291 / 377 101 / 156 a dose of not less than 1000 CCID50 per dose, live attenuated mumps virus present in a dose of not less than 5000 CCID50 per dose and live attenuated rubella virus present in a dose of not less than 1000 CCID50 per dose; 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); and b) a second container containing an aqueous solution selected from saline solution or water for injection (WFI) for reconstitution of the lyophilized (freeze-dried) vaccine composition.

[0439] According to some embodiments of the present description, the composition / formulation of the lyophilized / freeze-dried live attenuated MMR vaccine is in the form of a single-dose composition and is preservative-free.

[0440] In some embodiments of the present description, the composition / formulation of the freeze-dried / freeze-dried live attenuated MMR vaccine is in the form of a multidose composition.

[0441] In some embodiments of the present description, the composition of the lyophilized / freeze-dried live attenuated MMR vaccine is formulated for administration to a human individual or to children 2 years of age or younger.

[0442] In one embodiment of the present description, the composition of the freeze-dried / freeze-attenuated live MMR vaccine is safe, immunogenic, and induces persistence of protection.

[0443] Throughout this descriptive report, the word “includes”, Petition 870250085909, dated 09 / 23 / 2025, pp. 292 / 377 102 / 156 or variations such as “comprise” or “comprising”, will be understood as implying the inclusion of an element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps, and may mean “includes”, “including” and the like; “essentially consisting of” or “essentially consists” is also open, allowing the presence of more than what is mentioned, provided that the basic or new characteristics of what is mentioned are not altered by the presence of more than what is mentioned, but excludes prior art modalities.

[0444] The use of the expression “one or more” or “at least one” suggests the use of one or more elements, ingredients, or quantities, as used in the embodiment of the invention, to achieve one or more of the desired objectives or results. Although certain embodiments of the inventions have been described, these embodiments are presented only by way of example and are not intended to limit the scope of the inventions. Variations or modifications in the composition of this invention, within the scope of the invention, may occur to those skilled in the art when reviewing the description presented here. Such variations or modifications are perfectly within the spirit of this description.

[0445] The numerical values ​​provided for various physical parameters, dimensions and quantities are only approximate values ​​and it is anticipated that values ​​greater than the numerical value assigned to the physical parameters, dimensions and quantities fall within the scope of the invention, unless otherwise stated in the specification.

[0446] Although considerable emphasis has been placed here on the specific characteristics of the preferred modality, it will be appreciated that many additional characteristics could be added and that many Petition 870250085909, dated 09 / 23 / 2025, pp. 293 / 377 103 / 156 Changes may be made to the preferred embodiment without departing from the principles of the description. These and other changes to the preferred embodiment of the description will be evident to those skilled in the art from the description presented here, and it should be clearly understood that the descriptive matter above should be interpreted merely as an illustration of the description and not as a limitation. TECHNICAL ADVANTAGES

[0447] A method / process for manufacturing viral vaccines, relating to a composition / formulation of a live attenuated, freeze-dried / lyophilized viral vaccine. The monovalent, bivalent, or trivalent viral vaccine, comprising measles, mumps, and rubella antigens / immunogens as described above, offers several technical advantages, including, among others, the achievement of: 1. Improved, accessible, and safe large-scale manufacturing processes (encompassing cultivation, purification, and formulation stages). 2. It uses a minimum of animal-derived components, provides high viral yield, and ensures the preservation of the integrity / stability of the viral structure during manufacturing and storage. 3. pH, as one of the critical parameters of the cell growth process, is controlled by adequate ventilation to allow gas exchange and increase the buffering capacity of the cell medium. Adding an additional amount of NaHCO3 to the cell medium, which does not cause pH fluctuations, increases cell count and yield. 4. Use of vented MLTCF-10 with Corning vented caps (instead of Milex filter), resulting in slow dissociation of sodium bicarbonate. Petition 870250085909, dated 09 / 23 / 2025, pp. 294 / 377 104 / 156 5. The yield of mass measles vaccination is increased, i.e., by approximately > 50% (increase in batch yield from 8-9 million doses to 13-16 million doses) due to the ideal pH (7.2 to 7.8); ideal sodium bicarbonate concentration in the medium (1.0 to 2.5); low MOI (i.e., virus / cell ratio) (for mass measles and rubella vaccination 1:5 to 1:20 and for mass mumps vaccination 1:20 to 1:60); and multiple harvests. The increased yield of mass measles vaccination resulted in a change in the annual mass measles vaccination requirement from two facilities to one facility. 6. During the preparation of mass measles vaccination, the process of grouping the number of rolling vials during the collection phase, creating two groups of 250 rolling vials each, offers advantages such as fewer aseptic connections and aliquots from a well-mixed and homogenized pool, as well as fewer samples. 7. The Cell Cube used in the mass preparation of rubella vaccine offers advantages in terms of a high surface area to volume ratio and therefore better yields in a nearly closed system, which meets the principles of PAT. 8. The use of a 0.2 μm filter for filtering the stabilized virus during the preparation of mass rubella vaccine provides better assurance of sterility. 9. During the preparation of the mass mumps vaccine, the incubation of SPF eggs in an egg incubator with an expanded capacity of up to 2,400 eggs provides improvised production capacity. 10. Expanding mass production of mumps vaccines using MLTCF10 or CS10 and CF40 or CS40 provides qua Petition 870250085909, dated 09 / 23 / 2025, pp. 295 / 377 105 / 156 times the surface area for cultivation, with practically the same area occupied. 11. Use of recombinant trypsin (at least 95% purity, with a molecular weight of 23 to 25 kilodaltons; specific activity of 2500 USP units / mg; low endotoxin content) at an ideal pH (7.2 to 7.6). 12. Use of FBS irradiated with gamma rays. 13. Use of the ideal virus ratio: Stabilizer-I: Stabilizer-II (80:20:10). 14. The ideal pH range for the growth of MRC5 cells is 7.4 to 7.8, which improves trypsinization. Trypsinization is significantly improved in cultures maintained at physiological pH throughout the incubation period. Cells are easily detached, with little or no clumping compared to cells in acidic pH. This occurs because the cells are in the growth phase and the ideal pH for trypsin is in the range of 7.4 to 7.8. 15. A substantial amount of FBS can be saved. Currently, approximately 30.0 L of FBS are used per batch of measles. 7.0 to 8.0 L of FBS per batch (used as culture medium for medium change) can be saved. Annually, approximately 350.0 L of FBS can be saved. 16. Changing the medium is a possible way to increase cell yield, but it is not practical as it will increase costs. By avoiding changing the medium to MLTCF-10, the additional overhead in cleanrooms can be reduced, which will contribute to improved cGMP practices. 17. The volume of the final product (vaccine) in batches of 10 doses is reduced from 1.0 mL to 0.5 mL. 18. The consumption of Blind Vaccine (diluent used to dilute the volume during mixing) was reduced by approximately Petition 870250085909, dated 09 / 23 / 2025, pp. 296 / 377 106 / 156 30%. 19. The productivity of the bottling department will be increased. 20. The concept of a pH adjustment platform through gas exchange and the addition of extra NaHCO3 in a cellular medium may also be applicable to other mammalian cells, for example, Vero cells used for rotavirus and rabies vaccines. MODALITIES:

[0448] The present invention is illustrated in more detail by the following embodiments and combinations of embodiments, which result from the dependency references and corresponding links:

[0449] I. A method for producing a pool of clarified viruses, the method comprising: a. provide a cell line in a cell medium, a buffer, and a supplement; b. treat cells from the cell line with at least one enzyme; c. infect the cell line with a virus to form an infected cell line; d. Wash the infected cell line with a viral medium and buffer; e. collect the infected cell line from the medium to obtain a harvest; optionally, collect the infected cell line one or more times; f. add at least one stabilizer to the harvest; and g. clarify the harvest to obtain a clarified virus pool (CVP). optionally, where the cell line or the infected cell line in the exponential growth phase is - grown with additional ventilation or aeration, or Petition 870250085909, dated 09 / 23 / 2025, pp. 297 / 377 107 / 156 - supplied with buffer refills or - both.

[0450] II. The method as described in embodiment I, wherein the virus is selected from single-stranded RNA viruses, positive-sense, negative-sense, enveloped and non-enveloped, belonging to the families Picornaviridae, Caliciviridae, Togaviridae, Matonaviridae, Flaviviridae, Coronaviridae, Retroviridae, Filoviridae, Bunyaviridae, Rhabdoviridae, Orthomyxoviridae, Arenaviridae and Paramyxoviridae.

[0451] III. The method as described in either embodiment I to II, wherein the virus is a single-stranded, negative-sense, enveloped RNA virus belonging to the family Paramyxoviridae and a single-stranded, positive-sense, enveloped RNA virus belonging to the family Matonaviridae.

[0452] IV. The method as described in any of the embodiments I to III, wherein the virus is Measles Morbillivirus (measles virus), Mumps Orthorubulavirus (mumps virus) and Rubivirus rubellae (rubella virus).

[0453] V. The method as described in any of the embodiments I to IV, wherein the cell line is selected from animal cell line, mammalian cell line, avian cell line, insect cell line, human cell line, primary cell line, diploid cell line, continuous cell line, Rhesus monkey kidney cells (RhMK); Rabbit primary kidney cells; Human prepuce fibroblasts; Chicken embryo fibroblasts (CEF); Human squamous cell carcinoma cells (HEp-2); Human lung carcinoma cells (A549); Human cervical epithelial (HeLa); African green monkey renal epithelial (Vero); Human lung fibroblast (MRC-5); Human lung fibroblast (MRC-9); Mouse embryo fibroblast (NIH3T3); Mouse connective tissue fibroblast (L929); Chinese Hamster Ovarian Fibroblast (CHO); Fibroblasts Petition 870250085909, dated 09 / 23 / 2025, pp. 298 / 377 108 / 156 to Syrian Hamster Renal (BHK-21); Human Embryonic Renal Epithelial (HEK-293); Human Hepatic Epithelial (HepG2); Bovine Aortic Endothelial (BAE-1); Human Neuronal Neuroblastoma (SH-SY5Y); Mouse Myeloma Lymphoblast (NSO); Human Histiocytic 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); Canine kidney cells Madin Darby (MDCK); Human embryonic retinal cells (PER.C6); Human embryonic retinoblasts (HER.911); Murine non-secretory myeloma (Sp2.0); Epithelial cells of African green monkey renal origin (BSC-1 cells); Rhesus monkey renal epithelial cells (LLC-MK2 cells); Cercopithecus aethiops monkey renal cells (CV-1 cells); African green monkey renal fibroblast-like cells (COS cells); Crandell-Rees feline renal cells (CRFK cells); Rapidly Accelerating Fibrosarcoma cells (RAF cells); Normal rabbit renal epithelial cells (RK-13 ​​cells); C3H transformed mouse renal cells 1 (TCMK-1 cells); Porcine renal epithelial cells (LLCPK1 cells); Porcine renal cells (PK15 cells); Rabbit Renal Cell Line (LLC-RK1 cells), Non-secretory myeloma cell lines (NS-1 cells), Novel strain of human male diploid cells (TIG-1, TIG-7); Non-human primate diploid cell line (FRhL-2); Human fetal lung cells (IMR-90, IMR91); Human diploid lung fibroblasts and others.

[0454] VI. The method as described in any of the embodiments I to V, in which the cell line is selected from the Human Lung Fibroblast Cell Line (MRC-5) and the Cell Line Petition 870250085909, dated 09 / 23 / 2025, pp. 299 / 377 109 / 156 chicken embryo fibroblast cell (CEF).

[0455] VII. The method as described in any of the embodiments I to VI, wherein the enzyme is selected from trypsin, recombinant trypsin, dipase, collagenase, hyaluronidase, elastase, cysteine ​​protease, deoxyribonuclease I and chymotrypsin.

[0456] VIII. The method as described in any of the embodiments I to VII, wherein the cellular medium includes basal medium, enriched medium, selective and indicator medium, transport medium, storage medium, carbon sources or a combination thereof.

[0457] IX. The method as described in any of the embodiments I to VIII, wherein the supplement includes amino acids, cholesterol, proteins, lactoferrin, linoleic acid, yeast extracts, serums, antioxidants, vitamins, antibiotics, nutrients, trace elements, binding agents, extension factors or combinations thereof.

[0458] X. The method as described in any of embodiments I to IX, wherein the buffer is selected from NaHCOs, NaOH, NaCl, HEPES, PIPES, MES, phosphates, carbonates, Hank's, Earl's or combinations thereof.

[0459] XI. The method as described in any of embodiments I to X, wherein the viral medium for washing the infected cell line is selected from basal media, enriched media, selective and indicator media, transport media, storage media, carbon sources or a combination thereof.

[0460] XII. The method as described in any of the embodiments I to XI, in which washing of virally infected cell lines is carried out after infection with viruses having an MOI in the range of 1:5 to 1:60 and is followed by incubation at 30 to 40°C.

[0461] XIII. The method as described in any of embodiments I to XII, wherein at least one stabilizer includes at least one carbohydrate, at least one protein, at least one amino acid Petition 870250085909, dated 09 / 23 / 2025, pp. 300 / 377 110 / 156 or a combination thereof.

[0462] XIV. The method as described in any of the embodiments I to XIII, in which the clarification of the sample is carried out using filters, preferably the filters are of construction material selected from modified PVDF, cellulose acetate, polyethersulfone, polypropylene.

[0463] XV. The method as described in any of the embodiments I to XIV, in which the clarification of the sample is carried out using at least one filter with a pore size in the range of 0.1 to 10.0 μ.

[0464] XVI. The pool of clarified viruses (CVP) obtained by the method as described in any of the embodiments I to XV.

[0465] XVII. The method as described in any of embodiments I to XVI, for producing a pool of clarified measles virus, the method comprising: a. provide the cell line in a cell culture medium, buffer, and supplement; b. treat the cells of the cell line with the enzyme; c. infect the cell line with measles morbillivirus to form the measles-infected cell line; d. Wash the measles-infected cell line with the viral medium and buffer; e. collect the measles-infected cell line from the medium to obtain the harvest; optionally, collect the measles-infected cell line; f. add the stabilizer to the harvest; and g. clarify the sample to obtain the clarified measles virus pool (CVP). optionally, wherein the cell line or the measles-infected cell line in exponential growth phase is - grown with additional ventilation or aeration, or Petition 870250085909, dated 09 / 23 / 2025, pp. 301 / 377 111 / 156 - provided by the tampon readmission or - both.

[0466] XVIII. The method as described in embodiment XVII, wherein the cell line is the Human Lung Fibroblast cell line (MRC-5).

[0467] XIX. The method as described in either of the embodiments XVII or XVIII, wherein the enzyme is recombinant trypsin.

[0468] XX. The method as described in any of the modalities XVII to XIX, in which the cellular medium is the Minimum Essential Medium.

[0469] XXI. The method as described in any of the embodiments XVII to XX, in which the supplement includes glutamine and fetal bovine serum.

[0470] XXII. The method as described in embodiment XXI, in which fetal bovine serum is in the range of 5% to 15%.

[0471] XXIII. The method as described in any of the embodiments XVII to XXII, in which the viral medium is the Minimum Essential Medium.

[0472] XXIV. The method as described in any of the embodiments XVII to XXIII, wherein the buffer comprises sodium bicarbonate added to the viral medium and the buffer is in the range of 0.5 to 4.0 g / L to maintain the ideal pH of 6 to 8.

[0473] XXV. The method as described in any of the embodiments XVII to XXIV, wherein the measles virus includes the Edmonston strain, including the Schwartz, Edmonston-Zagreb, Moraten; CAM70; TD 97; Leningrad-16; AIK-C and Shanghai 191 (Ji-191) strains.

[0474] XXVI. A pool of clarified virus (CVP) for measles obtained by the method described in any of the embodiments XVII to XXV.

[0475] XXVII. The method as described in any of embodiments I to XVI for the production of a pool of clarified mumps virus, the method comprising: Petition 870250085909, dated 09 / 23 / 2025, pp. 302 / 377 112 / 156 a. provide the cell line in the cell culture medium, the buffer, and the supplement; b. treat the cells of the cell line with the enzyme; c. infect the cell line with the Mumps orthorubula virus to form a mumps-infected cell line; d. Wash the mumps-infected cell line with viral culture medium and buffer; e. collect the mumps-infected cell line in the culture medium to obtain the collection; optionally, collect the mumps-infected cell line; f. add the stabilizer to the collection; and g. clarify the collection to obtain the clarified mumps virus pool (CVP), optionally, in which the cell line or the mumps-infected cell line in the exponential growth phase is - grown with additional ventilation or aeration, or - supplied with tampon readmission or - both.

[0476] XXVIII. The method as described in embodiment XXVII, wherein the cell line is a Chicken Embryo Fibroblast Cell Line (CEF).

[0477] XXIX. The method as described in embodiments XXVII or XXVIII, wherein the enzyme is recombinant trypsin.

[0478] XXX. The method as described in any of the embodiments XXVII to XXIX, where the cellular medium is the Minimum Essential Medium.

[0479] XXXI. The method as described in any of the embodiments XXVII to XXX, wherein the supplement includes glutamine, fetal bovine serum and neomycin sulfate.

[0480] XXXII. The method as described in embodiment XXXI, in Petition 870250085909, dated 09 / 23 / 2025, pp. 303 / 377 113 / 156 that fetal bovine serum is in the range of 5% to 15%.

[0481] XXXIII. The method as described in any of the embodiments XXVII to XXXII, wherein the viral medium is the Minimum Essential Medium.

[0482] XXXIV. The method as described in any of the embodiments XXVII to XXXIII, wherein the buffer is sodium bicarbonate added to the viral medium and the buffer is in the range of 0.5 to 4.0 g / L to maintain the ideal pH of 6 to 8.

[0483] XXXV. The method as described in any of the embodiments XXVII to XXXIV, wherein the virus includes Jeryl-Lynn, RIT 4385, Leningrad-3, Leningrad-Zagreb (L-Zagreb), Urabe Am9, Hoshino strain, Torii strain and S79 Rubini strains.

[0484] XXXVI. A pool of clarified mumps virus (CVP) obtained by the method as described in any of the embodiments XXVII to XXXV.

[0485] XXXVII. The method as described in any of embodiments I to XVI for producing a pool of clarified rubella virus, the method comprising: a. provide the cell line in the cell culture medium and the supplement; b. treat the cells of the cell line with the enzyme; c. infect the cell line with Rubivirus rubellae to form a rubella-infected cell line; d. Wash the rubella-infected cell line with viral culture medium and buffer; e. collect the rubella-infected cell line in the culture medium to obtain the collection; optionally, collect the rubella-infected cell line; f. add the stabilizer to the harvest; and g. clarify the harvest to obtain a clarified virus pool Petition 870250085909, dated 09 / 23 / 2025, pp. 304 / 377 114 / 156 for rubella (CVP). optionally, wherein the cell line or the rubella-infected cell line in the exponential growth phase is - grown with additional ventilation or aeration, or - Provided for readmission of the tampon or - both.

[0486] XXXVIII. The method as described in embodiment XXXVII, wherein the cell line is the Human Lung Fibroblast cell line (MRC-5).

[0487] XXXIX. The method as described in embodiments XXXVII or XXXVIII, wherein the enzyme is recombinant trypsin.

[0488] XL. The method as described in any of the embodiments XXXVII to XXXIX, wherein the cellular medium is the Minimum Essential Medium.

[0489] XLI. The method as described in any of the embodiments XXXVII to XL, wherein the supplement includes glutamine, fetal bovine serum, neomycin sulfate or a combination thereof.

[0490] XLII. The method as described in embodiment XLI, wherein fetal bovine serum is in the range of 5% to 15%.

[0491] XLIII. The method as described in any of the embodiments XXXVII to XLII, wherein the viral medium is the Minimum Essential Medium.

[0492] XLIV. The method as described in any of the embodiments XXXVII to XLIII, wherein the buffer is sodium bicarbonate added to the viral medium and the buffer is in the range of 0.5 to 4 g / L to maintain the ideal pH of 6 to 8.

[0493] XLV. The method as described in any of the embodiments XXXVII to XLIV, wherein the virus includes the strains Wister RA 27 / 3, BRD-2, Matsuba, DCRB19, Takahashi, Matsuura and TO-336.

[0494] XLVI. A pool of clarified virus (CVP) for rubella obtained by the method as described in any of the embodiments Petition 870250085909, dated 09 / 23 / 2025, pp. 305 / 377 115 / 156 XXXVII to XLV.

[0495] XLVII. A method for obtaining a freeze-dried / lyophilized MMR immunogenic composition comprising at least one CVP selected from a CVP for measles, a CVP for mumps, a CVP for rubella or a combination thereof, the method comprising: To provide a measles vaccine (CVP) of modality XXVI or obtained by any of the modalities XVII to XXV, a mumps vaccine (CVP) of modality XXXVI or obtained by any of the modalities XXVII to XXXV, a rubella vaccine (CVP) of modality XLVI or obtained by any of the modalities XXXVII to XLV, or a combination thereof, mix the CVPs, followed by lyophilization of the mixed CVPs.

[0496] XLVIII. The method of modality XLVII, in which the method includes: a. Thaw at least one CVP (Combined Pulmonary Vital Sign) containing measles, mumps, rubella, or a combination of these viruses at 30 to 35 °C to obtain a thawed CVP; b. Mix the thawed CVP and the vaccine blindly to obtain a mixed solution; c. Clarify the mixed solution using a 0.45 μm filter to obtain a homogeneous volume; d) Aseptically fill the homogeneous volume into sterilized vials, followed by transferring the vials to a freeze dryer / lyophilizer. e) freeze-dry the vials containing the homogeneous volume.

[0497] XLIX. The method according to embodiment XLVIII, in which the freeze-drying / freeze-drying step comprises: Petition 870250085909, dated 09 / 23 / 2025, pp. 306 / 377 116 / 156 a) pre-freezing of the shelf, loading of trays containing vials of the MMR immunogenic composition onto the shelf; b) freezing; c) primary drying / sublimation d) secondary drying

[0498] L. An immunogenic composition of lyophilized / freeze-dried MMR obtained by the method described in embodiment XLIX, the composition comprising: a) at least one virus; b) a stabilizer comprising at least one carbohydrate, at least one amino acid and at least one hydrolyzed protein.

[0499] LI. The immunogenic composition of freeze-dried / lyophilized MMR, as described in embodiment L, comprising live attenuated measles virus present in a dose of not less than 1000 CCID50 per dose, live attenuated mumps virus present in a dose of not less than 5000 CCID50 per dose and live attenuated rubella virus present in a dose of not less than 1000 CCID50 per dose.

[0500] Lll. The immunogenic composition of freeze-dried / lyophilized MMR, as described in embodiment L, comprising at least one carbohydrate selected from a group consisting of natural carbohydrate, synthetic carbohydrate, monosaccharides, disaccharides, trisaccharides, oligosaccharides, reducing sugar, non-reducing sugar, sugar alcohols, polyol, polyhydroxylated compounds, chemically modified carbohydrates and glass transition facilitating agents, which include sucrose, mannitol, trehalose, mannose, raffinose, lactitol, lactobionic acid, glucose, maltulose, isomaltulose, maltose, lactose, sorbitol, dextrose, fructose, glycerol, sorbitol and fucose, and a combination Petition 870250085909, dated 09 / 23 / 2025, pages 307 / 377 117 / 156 nation of the same.

[0501] Llll. The immunogenic composition of freeze-dried / lyophilized MMR, as described in embodiment L, comprising at least one amino acid selected from a group consisting of tricine, leucine, isoleucine, 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.

[0502] LIV. The immunogenic composition of freeze-dried / lyophilized MMR, as described in embodiment L, comprising at least one hydrolyzed protein obtained by chemical, enzymatic or thermal hydrolysis of plant or animal proteins.

[0503] LV. The immunogenic composition of freeze-dried / lyophilized MMR, as described in embodiment L, comprising 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.

[0504] LVI. The immunogenic composition of freeze-dried / freeze-dried MMR, as described in embodiment L, wherein the composition comprises: a) at least one carbohydrate in the concentration range of 1 to 20% (w / v); b) at least one amino acid in the concentration range of 0.01 to 10% (w / v); c) at least one hydrolyzed protein in the concentration range of 0.1 to 10% (w / v);

[0505] LVII. The immunogenic composition of freeze-dried / lyophilized MMR, as described in embodiment LVI, in which at least one of the carbohydrates is sorbitol, present in a concentration Petition 870250085909, dated 09 / 23 / 2025, pp. 308 / 377 118 / 156 1 to 20% (w / v), 1 to 10% (w / v), preferably 3-6% (w / v).

[0506] LVIII. The immunogenic composition of freeze-dried / lyophilized MMR, as described in embodiment LVI, wherein at least one of the amino acids is 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).

[0507] LIX. The immunogenic composition of freeze-dried / lyophilized MMR, as described in embodiment LVI, wherein at least one of the hydrolyzed proteins is 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).

[0508] LX. The lyophilized / freeze-dried MMR immunogenic composition, as described in embodiment L, comprising an adjuvant selected from a group consisting of aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate and aluminum potassium sulfate, or a mixture thereof.

[0509] LXI. The immunogenic composition of freeze-dried / lyophilized MMR as described in embodiment L, comprising an immunostimulatory component selected from a group consisting of an oil-in-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, complete Freund's adjuvant, incomplete Freund's adjuvant, CRL-8300 adjuvant, muramyl dipeptide, TLR-4 agonists, flagellin, flagellin Petition 870250085909, dated 09 / 23 / 2025, pp. 309 / 377 119 / 156 in derivatives of gram-negative bacteria, TLR-5 agonists, flagellin fragments capable of binding to TLR-5 receptors, QS-21, ISCOMS, chitosan, saponin combination with sterols and lipids.

[0510] LXII. The immunogenic composition of freeze-dried / freeze-dried MMR, as described in embodiment L, comprising a pharmaceutically acceptable additive selected from a group consisting of a carrier, excipient, binder, isotonic agent, emulsifier and humectant.

[0511] LXIII. The immunogenic composition of freeze-dried / lyophilized MMR, as described in embodiment L, wherein the excipient is selected from a group consisting of salt, including NaCl, KCl, KH2PO4, Na2HPO4.2H2O, CaCl2 and MgCb; nonionic surfactant, including polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, oxtoxinol 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, and cyclodextrin.

[0512] LXIV. The lyophilized / freeze-dried MMR immunogenic composition, as described in embodiment L, wherein the lyophilized / freeze-dried viral vaccine composition is reconstituted with an aqueous solution selected from a group consisting of saline solution, buffer and WFI (water for injection).

[0513] LXV. The immunogenic composition of freeze-dried / lyophilized MMR, as described in embodiment LXIV, wherein the buffer is selected from a group consisting of sodium chloride, acetate, carbonate, citrate, lactate, gluconate, tartrate, phosphate buffer, saline solution, borate, histidine buffer, succinate buffer, HEPES, TRIS and citrate-phosphate. Petition 870250085909, dated 09 / 23 / 2025, pp. 310 / 377 120 / 156

[0514] LXVI. The lyophilized / freeze-dried MMR immunogenic composition, as described in embodiment LXIV, wherein the final pH of the reconstituted composition is in the range of pH 6.5 to 7.5.

[0515] LXVII. The immunogenic composition of freeze-dried / freeze-dried MMR, as described in any of the preceding embodiments L to LXVI, comprising: a) live attenuated measles virus present in a dose of not less than 1000 CCIDso per dose, live attenuated mumps virus present in a dose of not less than 5000 CCIDso per dose and live attenuated rubella virus present in a dose of not less than 1000 CCIDso 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).

[0516] LXVIII. The immunogenic composition of freeze-dried / freeze-dried MMR, as described in embodiment LXVII, comprising: a) live attenuated measles virus present in a dose of not less than 1000 CCIDso per dose, live attenuated mumps virus present in a dose of not less than 5000 CCIDso per dose and live attenuated rubella virus present in a dose of not less than 1000 CCIDso per dose; b) stabilizer comprising Petition 870250085909, dated 09 / 23 / 2025, pp. 311 / 377 121 / 156 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).

[0517] LXIX. The lyophilized / freeze-dried MMR immunogenic composition, as described in embodiment LXVIII, wherein the lyophilized viral vaccine composition is in the form of a single-dose composition or a multi-dose composition.

[0518] LXX. The lyophilized / freeze-dried MMR immunogenic composition, as described in embodiment LXIX, wherein the multi-dose composition additionally comprises a preservative.

[0519] LXXI. A kit comprising the lyophilized / freeze-dried MMR immunogenic composition, as described in embodiments LXVII and LXVIII, comprises: (a) a first container containing a freeze-dried viral vaccine composition, comprising said composition: live attenuated measles virus present in a dose of not less than 1000 CCIDso per dose, live attenuated mumps virus present in a dose of not less than 5000 CCIDso per dose and live attenuated rubella virus present in a dose of not less than 1000 CCIDso per dose; carbohydrate consisting of sorbitol present in a concentration of 1 to 10% (w / v); amino acid consisting of tricine present in a concentration of 0.1% to 2% (w / v), L-histidine Petition 870250085909, dated 09 / 23 / 2025, pp. 312 / 377 122 / 156 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 either saline or water for injection (WFI) for reconstitution of the lyophilized (freeze-dried) vaccine composition.

[0520] LXXII. A kit comprising the lyophilized / freeze-dried MMR immunogenic composition, as described in embodiments LXVII and LXVIII, comprises; (a) a first container containing a freeze-dried viral vaccine composition, wherein said composition comprises: live attenuated measles virus present in a dose of not less than 1000 CCIDso per dose, live attenuated mumps virus present in a dose of not less than 5000 CCID50 per dose and live attenuated rubella virus present in a dose of not less than 1000 CCIDso per dose; 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 hydrolyzed lactalbumin present at a concentration of 0.35% (w / v); and b) a second container containing an aqueous solution selected from saline solution or water for injection (WFI) for reconstitution of the freeze-dried vaccine composition. Petition 870250085909, dated 09 / 23 / 2025, pp. 313 / 377 123 / 156 EXAMPLES:

[0521] The preceding description of the modalities was provided for illustrative purposes and is not intended to limit the scope of the present description. Individual components of a specific modality are generally not limited to that specific modality, but are interchangeable. Such variations should not be considered a deviation from the present description, and all such modifications are considered within the scope of the present description.

[0522] The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those skilled in the art that the compositions and techniques described in the following examples represent techniques discovered by the inventor to work well in the practice of the invention and, therefore, may be regarded as preferred modes for its practice. The present description is described in more detail in the light of the following examples, which are presented for illustrative purposes only and should not be construed as limiting the scope of the description.

[0523] However, those skilled in the art should, in the light of the present description, understand that many alterations can be made to the specific embodiments described and still obtain a similar result without departing from the spirit and scope of the invention. EXAMPLE 1: A. Strains and their sources: The virus strains (measles, mumps, and rubella) and their sources are mentioned in Table 6 below. Table 6: Strains and their sources Measles virus strain, source of the measles vaccine, Edmonston-Zagreb Institute of Immunology, Zagreb, Croatia; Mumps virus strain, L-Zagreb Institute of Immunology, Zagreb, Croatia. Petition 870250085909, dated 09 / 23 / 2025, pp. 314 / 377 124 / 156 Rubella Virus Strain Source Rubella virus strain of the rubella vaccine Wister RA 27 / 3 Institute of Immunology, Zagreb, Croatia B. Cell lines and their sources: The cell lines used for virus growth are mentioned in Table 7 below. Table 7: Cell lineages and their sources Cell Line Source: Human Lung Fibroblast (MRC-5) cells at lower passage were obtained from NIBSC, UK. Chick Embryo Fibroblast (CEF) cells are prepared using 9-11 day old embryos of specific pathogen-free (SPF) chicken eggs. These SPF eggs are received from Lohmann, Germany, and Hy-Vac, USA. C. Ingredients selected for the viral vaccine manufacturing process (MMR vaccine): 1. Fetal bovine serum (FBS):

[0524] FBS is the most widely used basal medium supplement for in vitro cell culture. It contains very low levels of antibodies and a high concentration of growth factors, such as hormones, adhesion factors, and transport proteins. FBS effectively promotes and sustains cell growth at the low densities used in biological manufacturing. Serum also adds buffering capacity to the medium and binds or neutralizes toxic components. FBS is obtained from fetuses harvested from healthy animals at slaughterhouses. Manufacturer serum batches are pools of serum collected from various animals.

[0525] Because it is a material of animal origin, FBS presents an inherent risk of contamination by transmissible adventitious agents. In the case of serum obtained from slaughterhouses, it is generally not possible Petition 870250085909, dated 09 / 23 / 2025, pp. 315 / 377 125 / 156 vel demonstrate absence of bovine viral infections. Bovine serum can be contaminated by various bovine viruses. It is evident that each batch of serum should be tested for ubiquitous and known-risk viruses. The possibility of introduction and replication of adventitious agents during cell culture has long been recognized as a potential risk leading to viral contamination of the final product. There have been several cases where laboratory studies have provided evidence of the presence of adventitious agents in marketed vaccines. These risks of adventitious agents are normally mitigated by analyzing the absence of adventitious agents in bovine serum. Most regulatory bodies permit the use of animal-derived materials only when their use can be justified because there is no viable alternative. For this, each batch of serum must be tested for sterility and adventitious agents.Viral testing is typically performed in accordance with various regulatory guidelines. However, testing methods have their own limitations, and sometimes the adventitious contaminant may escape detection. Gamma irradiation:

[0526] Gamma irradiation is a very efficient and direct means of inactivating various types of viruses in FBS. Because FBS can be contaminated by adventitious viruses, gamma irradiation is, after routine quality control for virus detection, the best method to increase the safety of its use in the production of biological products. One of the reasons why the gamma irradiation method has been commonly used for the reduction of adventitious contaminants in serum is that it can be performed with serum in the original product containers. Furthermore, it is typically performed at low temperatures, thus maintaining the quality and performance of the serum unchanged. Both FBS irradiated with and without gamma irradiation are tested for suitability for cell growth and then used in Petition 870250085909, dated 09 / 23 / 2025, pp. 316 / 377 126 / 156 commercial production.

[0527] The FBS (from Australia / New Zealand) is irradiated with GAMMA irradiation used in this invention in the dose range of 20 to 50 KGy. 2. Recombinant trypsin:

[0528] Trypsin is a proteolytic enzyme (serine protease) used as a cell dispersal agent in tissue culture techniques. As such, it becomes an essential raw material for all tissue culture-based vaccines, such as measles, mumps, rubella, rabies, rotavirus, etc. Porcine trypsin is a widely used reagent in the manufacture of biological drugs. It is extracted from the porcine pancreas and, therefore, being a material of animal origin, presents an inherent risk of contamination by transmissible adventitious viruses. Approximately 55 species of porcine viruses from the human host range, from 17 different families, have been identified as potentially contaminating viruses. These risks from various adventitious agents are normally mitigated by testing porcine trypsin for the absence of foreign agents; however, testing methods have their own limitations, and sometimes contaminating adventitious agents may escape detection.

[0529] Regulatory guidelines now encourage the use of reagents free of animal components, such as recombinant trypsin, instead of animal-derived trypsin. Recombinant trypsin is a genetically modified protein, expressed in suitable microorganisms (E. coli, Pichia pastoris) and purified by high-performance liquid chromatography. The use of recombinant trypsin as an alternative to conventional porcine-derived trypsin can completely eliminate the risk of contamination by animal-derived reagents. Recombinant trypsin is supplied by two different suppliers (Biogenomics & Richcore (Laurus Bio)) for the production of clarified MMR virus pool or bulk MMR vaccine. Petition 870250085909, dated 09 / 23 / 2025, pp. 317 / 377 127 / 156 D. Cellular and Viral Media:

[0530] Cell lines were cultured in culture medium, buffer, and supplement. The cell lines were infected and the infected cell lines were cultured. The infected cell line was washed with viral medium and buffer.

[0531] The tables below (Tables 8 to 13) provide details on the cellular and viral media for the production of a clarified virus pool for measles, mumps, and rubella viruses. Table 8: Cellular media for the production of a clarified measles virus pool (10-liter batch) Serial No. Ingredient Unit of Measure Quantity / Batch or Lot Quantity per liter 1. MEM Powder Number of bottles (Equivalent to 10 liters of medium) 1 2. Glutamine gm 3.5 3. Sodium bicarbonate gm 15 4. Hydrochloric acid 1N ml 15 ml / As needed for pH adjustment. 5. Water for Injection (20°C-30°C) Lit qs to 10 lit. qs 6. Fetal Bovine Serum (FBS)* Lit 1.0 to 1.25 *The amount of FBS is added in a variable percentage from 10% to 12.5%, depending on the brand / batch number of the FBS and its growth-promoting properties. Table 9: Culture media for the production of a clarified mumps virus pool (10-liter batch size) Petition 870250085909, dated 09 / 23 / 2025, pp. 318 / 377 128 / 156 Serial No. Ingredient Unit of Measure Quantity / Batch or Lot Quantity per liter 1. MEM Powder Number of bottles (Equivalent to 10 liters of medium) 1 2. Glutamine gm 3.5 3. Sodium bicarbonate gm 15 4. Hydrochloric acid 1N ml 15 ml / As needed for pH adjustment. 5. Water for Injection (20°C-30°C) Lit qs for 10 lit. qs 6. Fetal Bovine Serum (FBS)* Lit 1.0 to 1.25 0.1 to 0.125 liter 7. Neomycin sulfate gm 0.55 *The amount of FBS is added in a variable percentage from 10% to 12.5%, depending on the FBS brand / batch and its growth-promoting properties. The exact batch size will vary based on the amount of FBS added per 10L of medium. Table 10: Culture media for producing a pool of clarified rubella virus (10-liter batch size) Serial No. Ingredient Unit of Measure Quantity / Batch or Lot Quantity per liter 1. MEM Powder Number of bottles (Equivalent to 10 liters of medium) 1 2. Glutamine gm 3.5 3. Sodium bicarbonate gm 15 4. Hydrochloric acid 1N ml 15 ml / As needed for pH adjustment. 5. Dextrose gm 5 6. Water for Injection (20-30°C) Lit qs for 10 lit. qs 7. Fetal Bovine Serum (FBS)* Lit 1.0 to 1.25 0.1 to 0.125 liter 8. Neomycin sulfate gm 0.55 The amount of FBS added varies from 10% to 12.5%, depending on the brand / batch number of FBS and its growth-promoting properties. Petition 870250085909, dated 09 / 23 / 2025, pp. 319 / 377 129 / 156 The exact batch size will vary based on the amount of FBS added per 10 Liters of medium. Table 11: Viral media for the production of a clarified measles virus pool (10-liter batch rate) Serial No. Ingredient Unit of Measure Quantity / Batch or Lot Quantity per liter 1. MEM Powder Bottle contents equivalent to 10 liters of viral medium 1 2. Sodium bicarbonate gm 7.5 3. Water for Injection 9-9.5 liters 4. Hydrochloric acid 1N* ml 7.5 ml / As needed for pH adjustment. 5. Water for Injection qs to 10 liters qs *The quantity is flexible depending on the activity of the ingredients. Table 12: Viral media for the production of a clarified mumps virus pool (10-liter batch size) Serial No. Ingredient Unit of Measure Quantity / Batch or Lot Quantity per liter 1. MEM Powder Bottle contents equivalent to 10 liters of viral medium 1 2. Sodium bicarbonate gm 7.5 3. Water for Injection 9-9.5 liters 4. Hydrochloric acid 1N* ml 7.5 ml / As needed for pH adjustment. 5. Water for Injection qs to 10 liters qs *The quantity is flexible depending on the activity and ingredients Table 13: Viral media for the production of a clarified rubella virus pool (10-liter batch size) Petition 870250085909, dated 09 / 23 / 2025, pp. 320 / 377 130 / 156 Serial No. Ingredient Unit of Measure Quantity / Batch or Lot Quantity per liter 1. MEM Powder Bottle contents equivalent to 10 liters of viral medium 1 2. Sodium bicarbonate gm 7.5 3. Dextrose gm 5 4. L-Glutamine gm 3.5 5. Water for Injection 9-9.5 liters 6. Hydrochloric acid 1N* ml 7.5 ml / As needed for pH adjustment. 7. Water for Injection qs to 10 liters qs *The quantity is flexible depending on the activity of the ingredients. EXAMPLE 2: FLOWCHART - MEASLES VACCINE PRODUCTION Reactivation of low-passage MRC-5 cells Φ Cell passage using tissue culture flasks Φ Preparation of cell factories / cell stacks for MRC-5 cells. Φ Trypsinization of cell factories / cell stacks and preparation of cell pools. Φ Cell distribution in roller bottles Φ Addition of the Seed Worker Virus to the rolls (infection) Φ Roller washing (MEM with FBS replaced using MEM without FBS) Petition 870250085909, dated 09 / 23 / 2025, pp. 321 / 377 131 / 156 Multiple harvest Φ Addition of stabilizers to the harvest Φ Clarification for preparing CVP (Clarified Virus Pool) Storage: below -20°C as a Measles Vaccine (Bulk Measles Vaccine) FLOWCHART: PRODUCTION OF THE MUMPS VACCINE Incubation of SPF eggs. Φ SPF egg candling to select healthy and fertile eggs. Φ Preparation of CEC (Chicken Embryo Culture) in cell factories / Cell stacks Φ Medium exchange applied to CEC in all culture vessels. Φ Addition of Working Seed Virus to CEC crops (infection) Φ CEC flushing (MEM with FBS replaced using MEM without FBS) Φ Multiple Harvest Φ Addition of stabilizers to the harvest Φ Clarification for preparing CVP (Clarified Virus Pool) Storage: below -20°C as a bulk mumps vaccine. FLOWCHART: PRODUCTION LOT OF THE RUPECTION VACCINE 870250085909, dated 09 / 23 / 2025, pages 322 / 377 132 / 156 BÉOLA Revivification of low-passage MRC-5 cells Φ Passage of MRC-5 cells using tissue culture flasks Preparation of MRC-5 cell factories Trypsinization of cell factories and preparation of cell pools. Φ Seeding cells in the Cell Cube system. Φ Large-scale production of MRC-5 cells in a cell cube system. Φ Adding the Working Seed Virus to Cell Cube modules (infection) Φ Cell Cube system flush (MEM with FBS replaced using MEM without FBS) Φ Multiple Harvest Φ Addition of stabilizers to the harvest Φ Clarification for preparing CVP (Clarified Virus Pool). Storage below -20°C as RUBELLA CVP (Bulk Rubella Vaccine). Lyophilization: Pools of Clarified Viruses (Measles, Mumps, and Rubella) Φ Petition 870250085909, dated 09 / 23 / 2025, pp. 323 / 377 133 / 156 Final Mass Φ Packaging Φ Freeze-drying Φ Final product (MMR vaccine - Immunogenic composition of live attenuated MMR, lyophilized / freeze-dried) EXAMPLE 3: Experiments / Optimizations performed during the production of the clarified measles virus pool A. Optimization of MRC-5 cell growth in Cell Factories (FBS Economy):

[0532] MRC-5 cells are cultured in cell growth medium containing Hank's MEM supplemented with 10% FBS, L-glutamine and 1.5 g / L sodium bicarbonate. The cultures are then incubated at 36°C ± 1°C for 4 to 6 days.

[0533] When MRC-5 cells are cultured in cell growth medium containing a CO2-bicarbonate buffering system, an initial alkaline shift occurs, with the pH higher (about 8.0) in about 2 to 8 hours, due to the absence of CO2 in the atmosphere, followed by progressive acidification of the medium, over a period of 48 to 96 hours, to a pH as low as 6.8; the rate and degree of these shifts vary with the seeding density and the metabolic activity of the cultured cells. The cultures eventually become “contact inhibited” and subsequent cells enter a stationary phase. Eagle, H. (1974) reported that maximum cell yield is obtained when MRC-5 cells are maintained at physiological or slightly higher pH throughout the incubation period (i.e., 7.4 to 7.8). At this pH, the initial contact inhibition of growth may not develop, and the cells may reach Petition 870250085909, dated 09 / 23 / 2025, pp. 324 / 377 134 / 156 gir population densities much higher than those normally observed.

[0534] This can be achieved by the following methods: 1. Supplementation with organic buffers:

[0535] To cultivate cells at a reasonably constant pH, the bicarbonate medium (26 mM) can be supplemented with non-gaseous buffers: “Hepes” 20-50 mM (N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid). It is more effective in maintaining physiological pH despite changes in CO2 concentrations. It is generally used in in vitro experiments. This represents a significant alteration in the process, therefore it was not logically used and performed. 2. Change of Environment / Perfusion of Environment:

[0536] Changes / Perfusion of the Medium had a profound effect on cellular metabolism, especially on DNA and protein synthesis. As the culture approached confluence, or the contact inhibition phenomenon, DNA, RNA, and protein synthesis was sequentially inhibited. Conversely, without a change of medium, sequential changes in macromolecular synthesis rates occurred as the culture approached confluence. DNA synthesis was affected first, followed by RNA synthesis, which declined very rapidly, and protein synthesis, which declined steadily but slowly. However, after a change of medium, there was a sequential stimulation of DNA, RNA, and protein synthesis in the same order in which they were inhibited. The inhibitory mechanism affected by cell clumping is obviously reversed by a change of medium.

[0537] To verify the possibility of changing the medium and whether changing the medium provides high cell yield, the following experiment was performed:

[0538] Spent medium of the cellular medium / cellular growth medium Petition 870250085909, dated 09 / 23 / 2025, pp. 325 / 377 135 / 156 of MLTCF-10 at 48 h and 120 h were analyzed for components such as residual glucose, lactic acid, amino acids (arginine, cystine, etc.), etc., and the percentage of utilization was calculated. The pH drop during cell growth was also determined. The results are summarized in Tables 14 and 15 below: Table 14: Use of change in cell growth medium Serial No. Component Actual Cell Growth Medium 48 h Expenditure 120 h Expenditure Cone, pg / ml Cone, pg / ml % Utilization Cone, pg / ml % Utilization (with 10% FBS) 1 Arginine 73.827 47.345 35.870 47.146 36.14 2 Cysteine ​​8.941 5.4745 38.771 2.984 66.63 3 Histidine 445.391 263.438 40.852 201.478 54.76 4 Isoleucine 48.542 27.072 44.230 25.839 46.77 5 Leucine 49.744 28.0465 43.618 28.316 43.08 6 Lysine 56.58 34.138 39.664 41.419 26.80 7 Methionine 14.086 9.176 34.857 9.262 34.25 8 Phenylalanine 30.038 18.926 36.993 24.157 19.58 9 Threonine 45.091 29.419 34.756 35.893 20.40 10 Tyrosine 27.388 24.138 11.867 26.044 4.91 11 Valine 48.435 30.785 36.441 31,878 34,18 12 Glutamic acid 12,6889 29,063 -129,043 69,66 -448,98 13 Serine 2,748 1,167 57,533 7,401 -169,32 14 Alanine 6,603 8,432 -27,700 35,893 -443,59 15 Proline 1,014 3,582 -253,254 14,33 -1313,21 16 Glycine 3,93 2,581 34,326 4,478 -13,94 17 Glucose 87 58 66,66 0 100% Petition 870250085909, dated 09 / 23 / 2025, pp. 326 / 377 136 / 156 Table 15: Change in pH pH profile Cell growth medium Time spent - 24 h Time spent - 48 h Time spent - 72 h Time spent - 96 h Time spent - 120 h 7.2 7.95 7.48 7.25 6.9 6.8

[0539] An amino acid would become growth-limiting if it were 100% utilized by the cells. It is observed that no nutrient, except glucose, was sufficiently utilized to become growth-limiting after 96 h of growth. It was observed that, in general, less than 50% of the amino acids were utilized by the cells and no amino acid was sufficiently utilized to become growth-limiting, even after 120 hours, which indicates that the change in medium alone did not increase cell yield.

[0540] Glucose is completely consumed by the cells in 120 hours. The glucose consumption pattern shows that utilization was completed after 72 hours. The pH fell below the physiological range between 72 and 96 hours. Under the conditions of this experiment, all amino acids were present in excess throughout the culture period. It appears that glucose, as well as acidic pH, may be the two limiting factors for growth. However, L-glutamine serves as an alternative carbon source, even if glucose is depleted from the medium, and therefore acidic pH remains the only limiting factor for growth. However, changing the medium may help supplement glucose, the instantaneous carbon source, and maintain the pH within the physiological range throughout the incubation period (96 hours), thus keeping the cells in the active metabolic stage.

[0541] Therefore, it is evident that changing the medium is neither necessary nor practical, as it will increase the cost and number of operations, materials, unnecessary movement of people in clean environments and, ultimately, the chance of contamination. Petition 870250085909, dated 09 / 23 / 2025, pages 327 / 377 137 / 156 3. Maximum cell yield without medium change

[0542] This can be achieved in two ways: a) Maintaining adequate gas exchange with the surrounding environment:

[0543] MRC-5 cells were cultured in cell growth medium containing Hank's MEM supplemented with 10% FBS and a sodium bicarbonate concentration of 2.0 g / L. The pH was adjusted to 6.9 to 7.1 by purging with CO2 gas before use. For MLTCF (Multilayered Tissue Culture Flask) -10 (CS-10, Corning or CF-10, Nunc's), the seeded cell count was 100 to 160 million cells and the temperature and incubation period were 36 ± 1 °C and 5 to 7 days, respectively. The MLTCF has two openings (0.2 μm² millix filters, 50 mm in diameter). The cell yield obtained was 400 to 500 million cells. The usual pH variations observed during the incubation period range from 7.8 to 7.0 or less.

[0544] Eagle, H. (1974) reported that maximum cell yield is obtained when MRC-5 cells are maintained at physiological pH or slightly higher throughout the incubation period (i.e., 7.4 to 7.6).

[0545] An experiment was planned to achieve this objective. In this experiment, Corning CS-10 filters (MLTCF-10) were used. The original vent caps were used instead of the milex filters on both ports. These vent caps are standard 33 mm threaded caps having non-wettable membranes with 0.2 µm pores, sealed directly to the caps to allow gas exchange. The results were compared with routinely used MLTCF-10s having 0.2 µm (50 mm) milex vent filters. Cultures were started from the same inocula (100 to 120 million cells). During the first 24 to 48 hours, when pH 0 Petition 870250085909, dated 09 / 23 / 2025, pages 328 / 377 Since a pH of 138 / 156 was lower (7.0 to 7.2) for fixation, the openings were hermetically sealed with sterile tape / aluminum foil to prevent gas exchange. After 48 hours, the openings were opened by removing the tape / aluminum foil to allow gas exchange. The pH remained in the range of 7.4 to 7.6 during the remainder of the incubation period. Details of cell yield and pH are summarized in Table 16. Table 16: Comparison of Ventilation (Milex filters) with Ventilation Covers (Corning Inc.) Passage No. Seeding Date Seeding Count (106) by MLTCF10 Passage Date. Ventilation (Milex filters) on both ports. Ventilation covers (Corning Inc.) on both ports. % increase in cell yield. Cell count (10⁶) PH spent. Cell count (10⁶) PH spent. P27 17.06.13 120 24.06.13 550 6.98 850 7.66 54.5 P27 18.06.13 115 25.06.13 500 7.10 820 7.71 64.0 P28 24.06.13 120 01.07.13 520 7.05 880 7.68 69.2 P28 25.06.13 105 02.07.13 500 7.12 920 7.76 84.0 P29 01.07.13 110 08.07.13 485 6.90 815 7.63 68.0 P29 02.07.13 120 09.07.13 500 7.18 840 7.78 68.0 P30 08.07.13 115 15.07.13 490 7.10 710 7.71 45.0 P30 09.07.13 110 16.07.13 410 7.14 880 7.78 114.0

[0546] It is observed that in MLTCF-10 with Milex filters (routine process), the pH dropped below the physiological level, while in MLTCF-10 with vented lids, the pH was maintained in the range of 7.6 to 7.78. The MLTCF-10 vented with Corning Vented lids produced 45 to 114% more cells than the MLTCF-10 vented with Milex filters.

[0547] Therefore, it is evident from the above data that the vented lids provide adequate gas exchange, due to the slow dissociation of sodium bicarbonate to maintain the alkaline pH, in comparison Petition 870250085909, dated 09 / 23 / 2025, pp. 329 / 377 139 / 156 tion with vented lids with Milex filters routinely used.

[0548] In cultures with Milex filters, where the pH fell below the non-physiological range (below 7.1), the lower cell growth (count) cannot have been caused by simple nutrient depletion, since merely maintaining the pH in the range of 7.6 to 7.8 produced a higher cell count when vented lids are used for gas exchange.

[0549] Another important observation is that trypsinization is significantly improved in cultures maintained at physiological pH throughout the incubation period. The cells are easily detached, with little or no clumping compared to cells in acidic pH. This is because the cells are in the growth phase and the ideal pH for trypsin is in the range of 7.4 to 7.8.

[0550] It is important to note that for proper gas exchange, Corning MLTCFs are ideal as their port size is ideal (33 mm) compared to Nunc MLTCFs, as the latter has a very narrow port size (10 mm) with a 50 mm Milex vent filter. b) Increasing the buffering capacity of the Cell Growth Medium: by increasing the concentration of sodium bicarbonate:

[0551] The pH control mechanism of Cell Culture Media is based on the bicarbonate buffer system. When dissolved in water, sodium bicarbonate (NaHCO3) dissociates to form a sodium ion (Na+) and a bicarbonate ion (HCO3-). The latter reacts with H+ in solution to form carbonic acid (H2CO3), which dissociates into CO2 and H2O. These two reactions reach their respective equilibria. The CO2 in solution also reaches equilibrium with CO2 in the gas phase. (See Equation 1) As a result, increasing the concentration of CO2 in the gas phase increases the amount Petition 870250085909, dated 09 / 23 / 2025, pp. 330 / 377 140 / 156 of CO2 dissolved in the culture medium, in turn increasing the concentration of H2CO3 and decreasing the pH. In contrast, if the concentration of CO2 in the gas phase is reduced, the pH will increase due to the reverse reaction.

[0552] The amount of sodium bicarbonate (NaHCO3) in the medium determines the amount of CO2 that must be used to maintain the desired pH. Excess carbon dioxide generated by the cells will increase the level of dissolved carbon dioxide and decrease the pH of the solution. To maintain the desired pH, additional required bicarbonate must be added.

[0553] Currently, in MMR production, NaHCO3 at 1.5 g / L is being used in the Cell Medium (CM) used for cell passage. MLTCF-10 / CS-10 is seeded with approximately 150 to 225 million cells in 2.0 L of CM. After 48 hours, it is replenished with 2.0 L of fresh CM (bicarbonate at 1.5 g / L). The pH profile of Cf-10 with and without medium change is calculated according to Table 17. Table 17: Comparison of MLTCF-10 / CS-10 without medium change and CF-10 / CS-10 with medium change in 48 hours, considering the pH parameter. Time pH: MLTCF-10 / CS-10 Without Medium Change pH: MLTCF-10 / CS-10 with Medium Change in 48 h. 0 h (CM) 7.45 7.45 24 h 7.95 7.95 48 h 7.35 7.37 72 h 7.15 7.40 96 h 6.95 7.05 120 h 6.80 7.02 144 h 6.80 6.90 168 h 6.80 6.85

[0554] As cells reach the exponential growth phase, they become metabolically active to the maximum and each Petition 870250085909, dated 09 / 23 / 2025, pp. 331 / 377 141 / 156 cell produces its maximum output of carbon dioxide.

[0555] The excess carbon dioxide generated by the cells increases the level of dissolved carbon dioxide and decreases the pH of the medium. Therefore, the acidic pH, acting as a limiting factor for growth, was controlled and maintained at the desired pH (7.5 to 7.7) throughout the incubation period by adding sodium bicarbonate to the culture medium in the range of 2.0 to 2.5 g / L.

[0556] In this way, the maximum yield of MRC-5 cells can be achieved in MLTCF-10 / CS-10 without the need to change the medium, thus saving a substantial amount of FBS, the most expensive item used in the production of the measles vaccine. B. pH maintenance:

[0557] pH is one of the critical parameters in the viral vaccine manufacturing process. Exposure of the virus to extreme pH inactivates it. Measles virus (MV) has been reported to be most stable at pH 7.6, with progressive inactivation occurring on both sides. The physical stability of MV is highly compromised in an acidic environment. Even small changes in pH affect the native conformation of the virus (Black, 1959). The infectivity of MV also depends on the conformational stability of the viral proteins. An alkaline medium not only neutralizes the acid produced by infected cells but also increases virus yield and stability after release into the medium. Therefore, maintaining the ideal pH throughout the process is unavoidable to preserve its native conformation. Furthermore, an alkaline medium has been reported to suppress the production of defective particles. (Yoshino et al. Archives of Virology 31-38 © SpringerVerlag 1975).

[0558] Similarly, for MRC-5, being a human diploid cell line, maintaining the pH in the physiological range or slightly higher (7.4 to 7.6) is equally important, since, in this Petition 870250085909, dated 09 / 23 / 2025, pp. 332 / 377 At a pH of 142 / 156, the cells are healthier and the binding sites (CD-46) for the human immunodeficiency virus would be in a more favorable state for infection. Furthermore, it is known that exposing cells to low pH induces the "Early Contact Inhibition phenomenon," which affects cell growth and, consequently, viral yield.

[0559] During routine MV production, after infection, infected cells are incubated at 36 ± 1 °C for 40 to 48 hours until washing. At this stage, it has been observed that the pH generally falls below 7.2. This indicates that the buffering capacity of the culture medium (CM) is insufficient to handle the acid produced by the infected cells and cannot maintain the pH above physiological levels. Therefore, to increase buffering capacity, the CM used for Cell Pool preparation should be supplemented with additional Sodium Bicarbonate (NaHCO3). Currently, the NaHCO3 concentration is 1.5 g / L. During harvesting, as the harvesting interval is much shorter (maximum 24.0 hours), the NaHCO3 concentration (0.75 g / L) in the Viral Medium (VM) appears to be sufficient and therefore there is no need to change it.With all this thought process and considering the seeding density per roll (40 to 60 million), the MOI (i.e., the virus-to-cell ratio) (1:8 to 1:12) and the post-infection incubation period (40 to 48 hours), the CM used only for the preparation of the Cell Pool was supplemented with 1.8 to 2.0 g / L of NaHCO3. The results are tabulated in Table 18 and illustrated in Figure 1. Table 18: Effect of NaHCO3 on measles vaccine yield (millions of doses) NaHCO3 = 1.5 g / L NaHCO3 = 1.8 g / L MD Batch No. MD Batch No. 0660M02 9.96 0661M01 17.80 0660M03 8.63 0661M02 14.54 0660M04 7.53 0661M03 15.85 Petition 870250085909, dated 09 / 23 / 2025, pp. 333 / 377 143 / 156 NaHCO3 = 1.5 g / L NaHCCh = 1.8 g / L MD Batch No. MD Batch No. 0660M05 8.02 0661M04 16.90 0660M06 7.92 0661M05 15.42 0660M07 7.70 0661M06 16.12 0660M08 7.05 0661M07 15.91 0660M09 7.42 0661M08 15.67 0660M10 6.95 0661M09 15.39 0660M11 7.13 0661M10 14.80 0660M12 6.55 0661M11 16.25 Average 7.72 15.88

[0560] It is observed that increasing the concentration of NaHCO3 in the culture medium used for preparing the cell pool reinforced the buffering capacity of the medium. Before washing the infected culture flasks, the pH of the medium used was checked and found not to fall below 7.2, remaining between 7.4 and 7.6. Therefore, since MEM has low buffering capacity, the pH must be controlled to obtain optimal cell growth and viral production. EXAMPLE 4:

[0561] The following tables (Table 19) present the components and their concentrations present in the immunogenic composition of lyophilized / freeze-dried live attenuated MMR. Table 19: Components and their concentration ranges in the immunogenic composition of lyophilized / freeze-dried live attenuated MMR (Measles, Mumps and Rubella (MMR) vaccine for a 0.5 mL dose) Component Quantity Measles virus Not less than 1000 CCID50 / 0.5 mL dose Mumps virus Not less than 5000 CCID50 / 0.5 mL dose Petition 870250085909, dated 09 / 23 / 2025, pp. 334 / 377 144 / 156 Component Quantity Rubella Virus Not less than 1000 CCID50 / 0.5 mL dose Gelatin (Partially hydrolyzed) 0.1-5% D-sorbitol 1-10% Histidine 0.1-1% L-Alanine 0.01-1% Tricine 0.1-1% Arginine 0.1-5% Lactalbumin Hydrolysate 0.1-10% Minimum Essential Medium (MEM) Base Table 20: Components and their concentrations in the immunogenic composition of freeze-dried / freeze-attenuated live MMR vaccine (Measles, Mumps and Rubella (MMR) vaccine) for a 0.5 mL dose. Component Quantity Measles virus Not less than 1000 CCID50 / 0.5 mL dose Mumps virus Not less than 5000 CCID50 / 0.5 mL dose Rubella virus Not less than 1000 CCID50 / 0.5 mL dose Gelatin (Partially hydrolyzed) 2.5% D-sorbitol 5% Histidine 0.21% L-Alanine 0.1% Tricine 0.3% Arginine 1.6% Lactalbumin hydrolysate 0.35% Minimum Essential Medium (MEM) Base Petition 870250085909, dated 09 / 23 / 2025, pp. 335 / 377 145 / 156 EXAMPLE 5: Tests performed on the final batch of the MMR vaccine

[0562] The tests performed on the final mass of the Measles, Mumps and Rubella vaccine are mentioned below in Table 21 and the summarized results of the MMR vaccine tests (Final Mass - 1 Dose) are mentioned in Tables 22 to 24. Table 21: Tests performed on the final batch of the Measles, Mumps, and Rubella Vaccine. TEST REQUIREMENTS REFERENCE Description Yellowish Liquid. In-House Sterility Test A 10.0 ml sample of the final mass vaccine is tested for sterility by direct inoculation into each type of medium, i.e., fluid thioglycolate digestion medium and soy casein. The final mass meets the sterility test requirements. Specification: Direct Inoculation Results meet the sterility test requirements. Ph. Eur. 5th Edition 2005, 2.6.1. Test for Residual Animal Serum Protein (Bovine Serum Albumin) With BSA: Not more than 50 ng per single human dose. Specification: Residual protein content: Not more than 50 ng per single human dose. Ph. Eur. 5th Edition 2005, 2.7.1. Test for Mycoplasma A 20 ml sample of the final mass vaccine is tested by the direct inoculation method. The final mass sample meets the test requirements for Mycoplasma. Ph. Eur. 5th Edition 2005, 2.6.7.Viral content test Specification: Direct inoculation Negative In-house. Table 22: Summary of results from the vaccine trials against MMR (Final Mass - 1 Portion) Petition 870250085909, dated 09 / 23 / 2025, pp. 336 / 377 146 / 156 SUMMARY OF RESULTS FROM VACCINE TESTS AGAINST Measles, Mumps and Rubella (Final Mass - 1 Dose) NO. BATCH TESTS Description Sterility Virus Content Residual Animal Protein Mycoplasma Yellowish liquid No growth should be observed in any of the sterility media For information only Not more than 50 ng / 0.5 mL dose Negative 1 Yellowish liquid No growth observed Satisfactory 2,310 ng / dose Negative 2 Yellowish liquid No growth observed Compliant 1,960 ng / dose Negative EU839 Yellowish liquid No growth observed Compliant 3,480 ng / dose Negative EU840 Yellowish liquid No growth observed Compliant 10,000 ng / dose Negative EU841 Yellowish liquid No growth observed Compliant 5,560 ng / dose Negative EU842 Yellowish liquid No growth observed Compliant 4,470 ng / dose Negative EU843 Yellowish liquid No growth observed Compliant 6,610 ng / dose Negative EU844 Yellowish liquid No growth observed Compliant 6,430 ng / dose Negative Petition 870250085909, dated 09 / 23 / 2025, pp. 337 / 377 147 / 156 EU845 Yellowish liquid No growth observed Compliant 4,020 ng / dose Negative EU846 Yellowish liquid No growth observed Compliant 2,700 ng / dose Negative EU847 Yellowish liquid No growth observed Compliant 1,950 ng / dose Negative EU848 Yellowish liquid No growth observed Compliant 10,780 ng / dose Negative EU849 Yellowish liquid No growth observed Compliant 1,830 ng / dose Negative EU850 Yellowish liquid No growth observed Compliant 7,630 ng / dose Negative EU852 Yellowish liquid No growth observed Compliant 5,160 ng / dose Negative EU853 Yellowish liquid No growth observed Compliant 1,370 ng / dose Negative EU854 Yellowish liquid No growth observed Compliant 7,070 ng / dose Negative EU855 Yellowish liquid No growth observed Compliant 7,120 ng / dose Negative EU856 Yellowish liquid No growth observed Compliant 2,680 ng / dose Negative EU858 Yellowish liquid No growth observed Compliant 3,400 ng / dose Negative, Table 23: Summary of results from MMR vaccine trials (Final Mass - 1 Dose) Petition 870250085909, dated 09 / 23 / 2025, pp. 338 / 377 148 / 156 SUMMARY OF RESULTS FROM VACCINE TESTS AGAINST Measles, Mumps and Rubella (Final Mass - 1 Dose) NO. BATCH TESTS Description Sterility Viral Content Residual Animal Protein Mycoplasma Yellowish liquid No growth should be observed in any of the sterility media For information only Not more than 50 ng / 0.5 mL dose Negative EU916 Yellowish liquid No growth observed Compliant 6,770 ng / dose Negative EU917 Yellowish liquid No growth observed Compliant 7,970 ng / dose Negative EU918 Yellowish liquid No growth observed Compliant 9,700 ng / dose Negative EU919 Yellowish liquid No growth observed Compliant 6,750 ng / dose Negative EU920 Yellowish liquid No growth observed Compliant 8,340 ng / dose Negative EU921 Yellowish liquid No growth observed Compliant 8,340 ng / dose Negative EU922 Yellowish liquid No growth observed Compliant 8,670 ng / dose Negative EU923 Yellowish liquid No growth observed Compliant 8,770 ng / dose Negative Petition 870250085909, dated 09 / 23 / 2025, pp. 339 / 377 149 / 156 EU924 Yellowish liquid No growth observed Compliant 4,460 ng / dose Negative EU925 Yellowish liquid No growth observed Compliant 2,960 ng / dose Negative EU941 Yellowish liquid No growth observed Compliant 0,790 ng / dose Negative EU942 Yellowish liquid No growth observed Compliant 9,740 ng / dose Negative EU943 Yellowish liquid No growth observed Compliant 10,550 ng / dose Negative EU944 Yellowish liquid No growth observed Compliant 17,210 ng / dose Negative EY945 Yellowish liquid No growth observed Compliant 9,570 ng / dose Negative EU946 Yellowish liquid No growth observed Compliant 6,210 ng / dose Negative EU947 Yellowish liquid No growth observed Compliant 9,500 ng / dose Negative EU948 Yellowish liquid No growth observed Compliant 3,300 ng / dose Negative EU949 Yellowish liquid No growth observed Compliant 2,980 ng / dose Negative EU950 Yellowish liquid No growth observed Compliant 0.520 ng / dose Negative, Table 24: Summary of results from the vaccine trials against MMR (Final Mass - 1 Portion) Petition 870250085909, dated 09 / 23 / 2025, pp. 340 / 377 150 / 156 SUMMARY OF RESULTS OF MEASLES, MUMPS AND RUBELLA VACCINE TESTS (FINAL MASS - 1 DOSE) BATCH NO. TEST DESCRIPTION Description Sterility Viral Content Residual Animal Protein Mycoplasma Yellowish liquid No growth should be observed in any of the sterility media For information only No more than 50 ng / 0.5 mL dose Negative 1070 Yellowish liquid No growth observed Compliant 10,810 ng / dose Negative 1071 Yellowish liquid No growth observed Compliant 16,700 ng / dose Negative 1072 Yellowish liquid No growth observed Compliant 12,980 ng / dose Negative 1073 Yellowish liquid No growth observed Compliant 12,660 ng / dose Negative 1074 Yellowish liquid No growth observed Compliant 15,070 ng / dose Negative 1075 Yellowish liquid No growth observed Compliant 9,950 ng / dose Negative 1076 Yellowish liquid No growth observed Compliant 13,540 ng / dose Negative 1077 Yellowish liquid No growth observed Compliant 15,970 ng / dose Negative 1078 Yellowish liquid No growth observed Compliant 8,360 ng / dose Negative 1079 Yellowish liquid No growth observed Compliant 8,230 ng / dose Negative 1080 Yellowish liquid No growth observed Compliant 15.26 ng / dose Negative 1081 Yellowish liquid No growth observed Compliant 5,450 ng / dose Negative Petition 870250085909, dated 09 / 23 / 2025, pp. 341 / 377 151 / 156 1082 Yellowish liquid No growth observed Compliant 25,350 ng / dose Negative 1083 Yellowish liquid No growth observed Compliant 3,040 ng / dose Negative 1084 Yellowish liquid No growth observed Compliant 11,110 ng / dose Negative 1085 Yellowish liquid No growth observed Compliant 7,520 ng / dose Negative 1086 Yellowish liquid No growth observed Compliant 10,990 ng / dose Negative 1087 Yellowish liquid No growth observed Compliant 9,120 ng / dose Negative 1088 Yellowish liquid No growth observed Compliant 19,530 ng / dose Negative 1089 Yellowish liquid No growth observed Compliant 14,470 ng / dose Negative 1090 Liquid Yellowish. No growth observed. Compliant. 13,850 ng / dose. Negative. EXAMPLE 6: Clinical study of the Live Attenuated (Freeze-Dried) Measles, Mumps and Rubella Vaccine (MMR vaccine)

[0563] The clinical study of the Live Attenuated Measles, Mumps and Rubella (Freeze-Dried) Vaccine (MMR vaccine), as mentioned in Table 25, is conducted considering the seroconversion parameter and observing adverse effects to evaluate the immunogenicity and safety / reactogenicity of the Live Attenuated Measles, Mumps and Rubella (Freeze-Dried) Vaccine (MMR vaccine). Table 25: Clinical study of the Live Attenuated (Freeze-Dried) Measles, Mumps and Rubella Vaccine (MMR Vaccine) Petition 870250085909, dated 09 / 23 / 2025, pp. 342 / 377 152 / 156 Product and Dose | Administration Routine | Age Group | Seroconversion | Measles | Mumps | Rubella | SIIPL MMR Vaccine (0.5 ml) IM (intramuscular) | 9-12 months | 81.00% | 91.50% | 100.00% | SIIPL MMR Vaccine (0.5 ml) SC (subcutaneous) | 9-18 months | 74.30% | 90.50% | 94.50% | SIIPL MMR Vaccine (0.5 ml) SC (subcutaneous) | 15-18 months | 84.00% | 82.00% | 84.00% | SIIPL MMR Vaccine (0.5 ml) SC (subcutaneous) | 18-24 months | 88.50% | 91.20% | 98.20%

[0564] Seroconversion is the transition from the point of infection to the presence of viral antibodies (IgG) in the blood. Table 25 showed that IgG positivity after immunization increased in the range of 70% to 90% for measles virus, 80% to 92% for mumps virus, and 80% to 100% for rubella virus. No adverse effects were observed after immunization. This indicates that the SIIPL MMR vaccine (0.5 ml dose) is immunogenic and safe. EXAMPLE 7: Stability data for Live Attenuated Lyophilized MMR Vaccine at 2-8°C for 3, 6, 9, 12, 18, 24 and 30 months (M) Table 26: Batch A - Stability data Test Limits / Initial Specification 3M 6M 9M 12M 18M 24M 30M Description Friable whitish-yellow mass Compliant Compliant Compliant Compliant Compliant Compliant Compliant Compliant Compliant Compliant Particulate matter (> 10μ) Must be less than 6000 particles per container 152 particles / container 181 particles / container 185 particles / container 102 particles / container 169 particles / container 106 particles / container 195 particles / container 155 particles / container Petition 870250085909, dated 09 / 23 / 2025, pages 343 / 377 153 / 156 Particulate matter (> 25μ) Must be less than 600 particles per container 2 particles / container 4 particles / container 2 particles / container 1 particle / container 1 particle / container 1 particle / container 6 particles / container 2 particles / container Residual moisture content Must be less than 2.000% w / w 0.500% w / w 0.532% w / w 0.534% w / w 0.471% w / w 0.451% w / w 0.507% w / w 0.407% w / w 0.509% w / w Measles virus concentration NLT potency 3.0 Log10 CCID50 / 0.5 mL 3.783 Log10 CCID50 / 0.5 mL 3.792 Log10 CCID5 Mumps Virus Concentration NLT Potency 0 / 0.5 mL 3.708 Log10 CCID5 0 / 0.5 mL 3.667 Log10 CCID5 0 / 0.5 mL 3.625 Log10 CCID5 0 / 0.5 mL 3.542 Log10 CCID5 0 / 0.5 mL 3.333L og10 CCID5 0 / 0.5 mL 3.250 Log10 CCID5 0 / 0.5 mL Mumps Virus Concentration NLT Potency 3.7 Log10 CCID5 0 / 0.5 mL 4.617 Log10 CCID5 0 / 0.5 mL 4.583 Log10 CCID5 0 / 0.5 mL 4.542 Log10 CCID5 0 / 0.5 mL 4.542 Log10 CCID5 0 / 0.5 mL 4.625 Log10 CCID5 0 / 0.5 mL 4.542 Log10 CCID5 0 / 0.5 mL 4.417 Log10 CCID5 0 / 0.5 mL 4,333 Log10 CCID5 0 / 0.5 mL Rubella virus concentration NLT potency 3.0 Log10 CCID5 0 / 0.5 mL 3.883 Log10 CCID5 0 / 0.5 mL 3.875 Log10 CCID5 0 / 0.5 mL 3.917 Log10 CCID5 0 / 0.5 mL 3.917 Log10 CCID5 0 / 0.5 mL 3.958 Log10 CCID5 0 / 0.5 mL 3.958 Log10 CCID5 0 / 0.5 mL 3.667 Log10 CCID5 0 / 0.5 mL 3.667 Log10 CCID5 0 / 0.5 mL Table 27: Batch B - Stability data Limits Test / Initial Specification 3M 6M 9M 12M 18M 24M 30M Petition 870250085909, dated 09 / 23 / 2025, pp. 344 / 377 154 / 156 Description Friable whitish-yellow mass Compliant Compliant Compliant Compliant Compliant Compliant Compliant Compliant Compliant Compliant Compliant Particulate matter (> 10μ) Must be less than 6000 particles per container 239 particles / container 163 particles / container 338 particles / container 203 particles / container 306 particles / container 309 particles / container 282 particles / container 261 particles / container Particulate matter (> 25μ) Must be less than 600 particles per container 2 particles / container 5 particles / container 4 particles / container 7 particles / container 8 particles / container 3 particles / container 1 particle / container 0 particles / container Residual moisture content Must be less than 2.000% w / w 0.460% w / w 0.513% w / w 0.434% w / w 0.406% w / w 0.410% Measles virus concentration - NLT potency 3.0 Log10 CCID50 / 0.5 mL 3, w / w 0.539% w / w 0.417% w / w 0.404% w / w883 Log10 CCID 50 / 0.5 mL 3.792 Log10 CCID5 0 / 0.5 mL 3.792 Log10 CCID5 0 / 0.5 mL 3.625 Log10 CCID5 0 / 0.5 mL 3.667 Log10 CCID5 0 / 0.5 mL 3.417 Log10 CCID5 0 / 0.5 mL 3,500L og10 CCID5 0 / 0.5 mL 3,458 Log10 CCID5 0 / 0.5 mL, Petition 870250085909, dated 09 / 23 / 2025, pages 345 / 377 155 / 156 Mumps virus concentration - NLT potency 3.7 Log10 CCID50 / 0.5 mL 4.717 Log10 CCID50 / 0.5 mL 4.708 Log10 CCID50 / 0.5 mL 4.625 Log10 CCID50 / 0.5 mL 4.625 Log10 CCID50 / 0.5 mL 4.500 Log10 CCID50 / 0.5 mL 4.292 Log10 CCID50 / 0.5 mL 4.333 Log10 CCID50 / 0.5 mL 4.417 Log10 CCID50 / 0.5 mL Rubella virus concentration NLT potency 3.0 Log10 CCID50 / 0.5 mL 3.983 Log10 CCID50 / 0.5 mL 3.917 Log10 CCID5 0 / 0.5 mL 3.958 Log10 CCID5 0 / 0.5 mL 3.875 Log10 CCID5 0 / 0.5 mL 3.917 Log10 CCID5 0 / 0.5 mL 4.000 Log10 CCID5 0 / 0.5 mL 3.875 Log10 CCID5 0 / 0.5 mL 3.792 Log10 CCID5 0 / 0.5 mL Table 28: Batch C - Stability Data Test Limits / Initial Specification 3M 6M 9M 12M 18M 24M 30M Description Friable whitish-yellow mass Compliant Compliant Compliant Compliant Compliant Compliant Compliant Compliant Compliant Compliant Particulate matter (> 10μ) Must be less than 6000 particles per container 224 particles / container 210 particles / container 267 particles / container 210 particles / container 446 particles / container 436 particles / container 201 particles / container 215 particles / container Particulate matter (> 25μ) Must be less than 600 particles per container 3 particles / container 3 particles / container 1 particle / container 3 particles / container 9 particles / container 11 particles / container 3 particles / container 3 particles / container Petition 870250085909, dated 09 / 23 / 2025, pages 346 / 377 156 / 156 Residual moisture content Must be less than 2.000% w / w 0.426% w / w 0.522% w / w 0.420% w / w 0.431% w / w 0.459% w / w 0.457% w / w 0.448% w / w 0.382% w / w Measles virus concentration Potency NLT 3.0 Log 10 CCID50 / 0.5 mL 3.683 Log 10 CCID5 0 / 0.5 mL 3.625 Log 10 CCID5 0 / 0.5 mL 3.875 Log 10 CCID5 0 / 0.5 mL 3.583 Log 10 CCID5 0 / 0.5 mL 3.542 Log 10 CCID5 0 / 0.5 Mumps Virus Concentration Potency NLT 3.7 Log 10 CCID5 0 / 0.5 mL 4.583 Log 10 CCID5 0 / 0.5 mL 4.625 Log 10 CCID5 0 / 0.5 mL 4.667 Log 10 CCID5 0 / 0.5 mL 4.583 Log 10 CCID5 0 / 0.5 mL 4.583 Log 10 CCID5 0 / 0.5 mL 4.542 Log 10 CCID5 0 / 0.5 mL 4.375 Log 10 CCID5 0 / 0.5 mL 4.167 Log 10 CCID5 0 / 0.5 mL Rubella virus concentration NLT potency 3.0 Log 10 CCID5 0 / 0.5 mL 3.767 Log 10 CCID5 0 / 0.5 mL 3.708 Log 10 CCID5 0 / 0.5 mL 3.708 Log 10 CCID5 0 / 0.5 mL 3.833 Log 10 CCID5 0 / 0.5 mL 3.792 Log 10 CCID5 0 / 0.5 mL 3.708 Log 10 CCID5 0 / 0.5 mL 3.667 Log 10 CCID5 0 / 0.5 mL 3.542 Log 10 CCID5 0 / 0.5 mL,

[0565] In Tables 26, 27 and 28, it was found that the Live Attenuated Lyophilized MMR Vaccine is stable at 2-8 °C for 3, 6, 9, 12, 18, 24, 30 months (M). Petition 870250085909, dated 09 / 23 / 2025, pages 347 / 377

Claims

1 / 18 CLAIMS 1. A method for producing a clarified virus pool, characterized in that it comprises: a. providing a cell line in a cell culture medium, 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 the infected cell line with a viral culture medium and the buffer; e. collecting the infected cell line in the medium to obtain a harvest; optionally, collecting 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 the exponential growth phase is - cultured with ventilation or additional aeration, or - provided with buffer re-additions, or - both.

2. Method, according to claim 1, characterized in that the virus is selected from single-stranded RNA viruses, positive-sense, negative-sense, enveloped and non-enveloped, belonging to the families Picornaviridae, Caliciviridae, Togaviridae, Matonaviridae, Flaviviridae, Coronaviridae, Retroviridae, Filoviridae, Bunyaviridae, Rhabdoviridae, Orthomyxoviridae, Arenaviridae and Paramyxoviridae.

3. Method, according to claim 1 or 2, characterized by the fact that the virus is composed of 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. A method according to any one of claims 1 to 3, characterized in that the virus is Measles morbillivirus (measles virus), Mumps orthorubulavirus (mumps virus), and Rubivirus rubellae (rubella virus).

5. Method, according to any one of claims 1 to 4, characterized in that the cell line is selected from animal cell line, mammalian cell line, bird cell line, insect cell line, human cell line, primary cell line, diploid cell line, continuous cell line, Rhesus monkey kidney cells (RhMK); Rabbit primary kidney cells; Human prepuce fibroblasts; Chicken embryo fibroblasts (CEF); Human squamous cell carcinoma cells (HEp-2); Human lung carcinoma cells (A549); Human cervical epithelial cells (HeLa); African green monkey renal epithelial cells (Vero); Human lung fibroblast (MRC-5); Human lung fibroblast (MRC-9); Mouse embryo fibroblast (NIH3T3); Mouse connective tissue fibroblast (L929); Chinese Hamster Ovarian Fibroblast (CHO); Syrian Hamster Renal Fibroblast (BHK-21); Human Embryonic Renal Epithelial Cell (HEK-293); Human Hepatic Epithelial Cell (HepG2);Bovine Aortic Endothelial Cell (BAE-1); Human Neuronal Neuroblastoma (SH-SY5Y); Mouse Myeloma Lymphoblast (NSO); Human Histiocytic 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); Petition 870250085909, dated 09 / 23 / 2025, page 349 / 377 3 / 18 Human embryonic lung cells (W1-38); Canine Madin Darby kidney cells (MDCK); Human embryonic retinal cells (PER.C6); Human embryonic retinoblasts (HER.911); Murine non-secretory myeloma (Sp2.0); African green monkey renal epithelial cells (BSC-1 cells); Rhesus monkey renal epithelial cells (LLC-MK2 cells); Cercopithecus aethiops monkey renal cells (CV-1 cells);African green monkey renal fibroblast-like cells (COS cells); Crandell-Rees feline renal cells (CRFK cells); Rapidly Accelerating Fibrosarcoma cells (RAF cells); Normal rabbit renal epithelial cells (RK-13 ​​cells); C3H-transformed mouse renal cells 1 (TCMK-1 cells); Porcine renal epithelial cells (LLC-PK1 cells); Porcine renal cells (PK15 cells); Rabbit renal cell line (LLC-RK1 cells); Non-secretory myeloma cell lines (NS-1 cells); Novel strain of human male diploid cells (TIG-1, TIG-7); Non-human primate diploid cell line (FRhL-2); Human fetal lung cells (IMR-90, IMR-91); human diploid lung fibroblasts and others.

6. Method, according to any one of claims 1 to 5, characterized in that the cell line is selected from Human Lung Fibroblast Cell Line (MRC-5) and Chicken Embryo Fibroblast Cell Line (CEF).

7. A method according to any one of claims 1 to 6, characterized in that the enzyme is selected from trypsin, recombinant trypsin, dipase, collagenase, hyaluronidase, elastase, cysteine ​​protease, deoxyribonuclease I, and chymotrypsin.

8. Method, according to any one of claims 1 to 7, characterized in that the cellular medium includes basal medium, enriched medium, selective and indicator medium, transport medium, storage medium, carbon sources or a combination thereof.

9. A method, according to any one of claims 1 to 8, characterized in that the supplement includes amino acids, cholesterol, proteins, lactoferrin, linoleic acid, yeast extracts, serums, antioxidants, vitamins, antibiotics, nutrients, trace elements, binding agents, extending factors, or combinations thereof.

10. A method according to any one of claims 1 to 9, characterized in that the buffer is selected from NaHCO3, NaOH, NaCl, HEPES, PIPES, MES, phosphates, carbonates, Hank's, Earl's, or combinations thereof.

11. A method, according to any one of claims 1 to 10, characterized in that the viral medium for washing the infected cell line is selected from basal media, enriched media, selective and indicator media, transport media, storage media, carbon sources, or combinations thereof.

12. Method, according to any one of claims 1 to 11, characterized in that the washing of virally infected cell lines is carried out after infection with viruses having an MOI in the range of 1:5 to 1:60 and is followed by incubation at 30 to 40°C.

13. A method according to any one of claims 1 to 12, characterized in that at least one stabilizer includes at least one carbohydrate, at least one protein, at least one amino acid, or a combination thereof.

14. Method, according to any one of claims 1 to 13, characterized in that the clarification of the harvest is carried out using filters, preferably the filters are of construction material selected from modified PVDF, cellulose acetate, polyethersulfone, polypropylene.

15. Method, according to any one of claims 1 to 14, characterized in that the clarification of the harvest is carried out using at least one filter with a pore size in the range of 0.1 to 10.0 μm.

16. Pool of clarified viruses (CVP), characterized in that it is obtained by the method as defined in any one of claims 1 to 15.

17. A method, according to any one of claims 1 to 16, for producing a pool of clarified measles virus, characterized in that it comprises: a. providing the cell line in a cell culture medium, buffer and supplement; b. treating the cell line cells with the enzyme; c. infecting the cell line with Measles morbillivirus to form the measles-infected cell line; d. washing the measles-infected cell line with the viral culture medium and buffer; e. collecting the measles-infected cell line in the culture medium to obtain the harvest; optionally, collecting the measles-infected cell line; f. adding the stabilizer to the harvest; and g. clarifying the harvest to obtain the pool of clarified measles virus (CVP).Optionally, wherein the cell line or the measles-infected cell line in exponential growth phase is - cultured with additional ventilation or aeration, or - provided with buffer readmission, or - both. Petition 870250085909, dated 09 / 23 / 2025, pp. 352 / 377 6 / 18.

18. Method according to claim 17, characterized in that the cell line is a Human Lung Fibroblast cell line (MRC-5).

19. Method according to claim 17 or 18, characterized in that the enzyme is recombinant trypsin.

20. Method, according to any one of claims 17 to 19, characterized in that the cellular medium is the Minimum Essential Medium.

21. Method, according to any one of claims 17 to 20, characterized in that the supplement includes glutamine and fetal bovine serum.

22. Method according to claim 21, characterized in that the fetal bovine serum is in the range of 5% to 15%.

23. Method, according to any one of claims 17 to 22, characterized in that the viral medium is the Minimum Essential Medium.

24. Method, according to any one of claims 17 to 23, characterized in that the buffer comprises sodium bicarbonate added to the viral medium and the buffer is in the range of 0.5 to 4.0 g / L to maintain the ideal pH of 6 to 8.

25. Method, according to any one of claims 17 to 24, characterized in that the measles virus includes the Edmonston strain, including the Schwartz, Edmonston-Zagreb, Moraten strains; CAM-70; TD 97; Leningrad-16; AIK-C strain and Shanghai 191 (Ji-191) strains.

26. Pool of clarified virus (CVP) for measles, characterized in that it is obtained by the method as defined in any one of claims 17 to 25.

27. Method, according to any one of claims 1 to 16, for the production of a clarified virus pool for mumps, characterized in that it comprises: a. providing the cell line in cell culture medium, buffer and supplement; b. treating the cell line cells with the enzyme; c. infecting the cell line with Mumps ortorubula virus to form a mumps-infected cell line; d. washing the mumps-infected cell line with viral culture medium and buffer; e. collecting the mumps-infected cell line in culture medium to obtain the collection; optionally, collecting the mumps-infected cell line; f. adding stabilizer to the collection; and g. clarifying the collection to obtain the clarified mumps virus pool (CVP).Optionally, where the cell line or the mumps-infected cell line in the exponential growth phase is either - cultured with additional ventilation or aeration, or - provided with buffer readmission, or - both.

28. Method according to claim 27, characterized in that the cell line is a chicken embryo fibroblast cell line (CEF).

29. Method according to claim 27 or 28, characterized in that the enzyme is recombinant trypsin.

30. Method, according to any one of claims 27 to 29, characterized in that the cellular medium is the Minimum Essential Medium.

31. Method, according to any one of claims 27 to 30, characterized in that the supplement includes glutamine, fetal bovine serum, and neomycin sulfate. Petition 870250085909, dated 09 / 23 / 2025, pp. 354 / 377 8 / 18 32. Method according to claim 31, characterized in that the fetal bovine serum is in the range of 5% to 15%.

33. Method, according to any one of claims 27 to 32, characterized in that the viral medium is the Minimum Essential Medium.

34. Method, according to any one of claims 27 to 33, characterized in that the buffer is sodium bicarbonate added to the viral medium and the buffer is in the range of 0.5 to 4.0 g / L to maintain the ideal pH of 6 to 8.

35. Method, according to any one of claims 27 to 34, characterized in that the virus includes Jeryl-Lynn, RIT 4385, Leningrad-3, Leningrad-Zagreb (L-Zagreb), Urabe Am9, Hoshino strain, Torii strain and S79 Rubini strains.

36. Pool of clarified mumps virus (CVP), characterized in that it is obtained by the method as defined in any one of claims 27 to 35.

37. A method, according to any one of claims 1 to 16, for producing a clarified rubella virus pool, characterized in that it comprises: a. providing the cell line in the cell medium and supplement; b. treating the cell line cells with the enzyme; c. infecting the cell line with Rubivirus rubellae to form a rubella-infected cell line; d. washing the rubella-infected cell line with the viral medium and buffer; e. collecting the rubella-infected cell line in the medium to obtain the harvest; optionally, collecting the rubella-infected cell line; f. adding the stabilizer to the harvest; and g. clarifying the harvest to obtain the clarified rubella virus pool (CVP).Optionally, the cell line or the rubella-infected cell line in the exponential growth phase is either - cultured with additional ventilation or aeration, or - provided with buffer readmission, or - both.

38. Method according to claim 37, characterized in that the cell line is a Human Lung Fibroblast cell line (MRC-5).

39. Method according to claim 37 or 38, characterized in that the enzyme is recombinant trypsin.

40. Method, according to any one of claims 37 to 39, characterized in that the cellular medium is the Minimum Essential Medium.

41. A method, according to any one of claims 37 to 40, characterized in that the supplement includes glutamine, fetal bovine serum, neomycin sulfate, or a combination thereof.

42. Method according to claim 41, characterized in that the fetal bovine serum is in the range of 5% to 15%.

43. A method, according to any one of claims 37 to 42, characterized in that the viral medium is the Minimum Essential Medium.

44. Method, according to any one of claims 37 to 43, characterized in that the buffer is sodium bicarbonate added to the viral medium and the buffer is in the range of 0.5 to 4 g / L to maintain the ideal pH of 6 to 8.

45. Method, according to any one of claims 37 to 44, characterized in that the virus includes the strain Wis Petition 870250085909, dated 09 / 23 / 2025, page 356 / 377 10 / 18 ter RA 27 / 3, the strain BRD-2, the strains Matsuba, DCRB19, Takahashi, Matsuura and TO-336.

46. ​​Pool of clarified rubella virus (CVP), characterized in that it is obtained by the method as defined in any one of claims 37 to 45.

47. Method for obtaining a freeze-dried / lyophilized MMR immunogenic composition comprising at least one PVC selected from a measles PVC, a mumps PVC, a rubella PVC or a combination thereof, characterized in that it comprises: providing a measles PVC as defined in claim 26 or obtained as defined in any of claims 17 to 25, a mumps PVC as defined in claim 36, or obtained as defined in any of claims 27 to 35, a rubella PVC as defined in claim 46, or obtained as defined in any of claims 37 to 45, or a combination thereof, mixing the PVCs, followed by freeze-drying the mixed PVCs.

48. Method according to claim 47, characterized in that it includes: a) thawing at least one CVP of measles, mumps, rubella virus or a combination thereof at 30 to 35 °C to obtain the thawed CVP; b) blindly mixing the thawed CVP and the vaccine to obtain a mixed solution; c) clarifying the mixed solution through a 0.45 μm filter to obtain a homogeneous volume; d) aseptically filling the homogeneous volume into sterilized vials, followed by transferring the vials to a freeze-dryer; e) freeze-drying the vials containing the homogeneous volume.

49. Method according to claim 48, characterized in that the freeze-drying / freeze-drying step comprises: a) pre-freezing the shelf, loading the trays containing the MMR immunogenic composition vials onto the shelf; b) freezing; c) primary drying / sublimation and d) secondary drying.

50. Immunogenic composition of lyophilized / freeze-dried MMR obtained by the method as defined in claim 49, characterized in that it comprises: a) at least one virus; b) a stabilizer comprising at least one carbohydrate, at least one amino acid and at least one hydrolyzed protein.

51. Lyophilized / freeze-dried MMR immunogenic composition according to claim 50, characterized in that it comprises live attenuated measles virus present in a dose of not less than 1000 CCIDso per dose, live attenuated mumps virus present in a dose of not less than 5000 CCIDso per dose and live attenuated rubella virus present in a dose of not less than 1000 CCIDso per dose.

52. Immunogenic composition of lyophilized / freeze-dried MMR, according to claim 50, characterized in that it comprises at least one carbohydrate selected from a group consisting of natural carbohydrate, synthetic carbohydrate, monosaccharides, disaccharides, trisaccharides, oligosaccharides, reducing sugar, non-reducing sugar, sugar alcohols, polyol, compos Petition 870250085909, dated 09 / 23 / 2025, page. 358 / 377 12 / 18 polyhydroxylated compounds, chemically modified carbohydrates and glass transition facilitators, which include sucrose, mannitol, trehalose, mannose, raffinose, lactitol, lactobionic acid, glucose, maltulose, isomaltulose, maltose, lactose, sorbitol, dextrose, fructose, glycerol, sorbitol and fucose, and a combination thereof.

53. Immunogenic composition of lyophilized / freeze-dried MMR, according to claim 50, characterized in that it comprises at least one amino acid selected from a group consisting of tricine, leucine, isoleucine, 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. Immunogenic composition of lyophilized / freeze-dried MMR, according to claim 50, characterized in that it comprises at least one hydrolyzed protein obtained by chemical, enzymatic or thermal hydrolysis of protein of vegetable or animal origin.

55. Immunogenic composition of lyophilized / freeze-dried MMR, according to claim 50, characterized in that it comprises 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. Immunogenic composition of lyophilized / freeze-dried MMR, according to claim 50, characterized in that it comprises: d) at least one carbohydrate in the concentration range of 1 to 20% (w / v); e) at least one amino acid in the concentration range of 0.01 to 10% (w / v); f) at least one hydrolyzed protein in the concentration range of 0.1 to 10% (w / v) 57. Immunogenic composition of lyophilized / freeze-dried MMR, according to claim 56, characterized in that at least one of the carbohydrates is sorbitol, present in a concentration of 1 to 20% (w / v), 1 to 10% (w / v), preferably 3 to 6% (w / v).

58. Immunogenic composition of lyophilized / freeze-dried MMR, according to claim 56, characterized in that at least one of the amino acids is selected from a group consisting of tricine, present in a concentration of 0.1% to 2% (w / v), L-histidine, present in a concentration of 0.1% to 2% (w / v), L-alanine, present in a concentration of 0.01% to 1% (w / v) and L-arginine hydrochloride, present in a concentration of 0.1% to 5% (w / v).

59. Immunogenic composition of lyophilized / freeze-dried MMR, according to claim 56, characterized in that at least one of the hydrolyzed proteins is selected from a group consisting of gelatin present in a concentration of 0.1% to 5% (w / v) and lactalbumin hydrolysate present in a concentration of 0.1% to 2% (w / v).

60. Lyophilized / freeze-dried MMR immunogenic composition according to claim 50, characterized in that it comprises an adjuvant selected from a group consisting of aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate and aluminum potassium sulfate or a mixture thereof.

61. Immunogenic composition of lyophilized / freeze-dried MMR, according to claim 50, characterized in Petition 870250085909, dated 09 / 23 / 2025, page 1. 360 / 377 14 / 18 because it comprises an immunostimulatory component selected from a group consisting of an oil-in-water emulsion, MF-59, a liposome, a lipopolysaccharide, a saponin, lipid A, lipid A derivatives, monophosphoryl lipid A, deacylated monophosphoryl lipid A, AS01, AS03, an oligonucleotide, an oligonucleotide comprising at least one unmethylated CpG and / or a liposome, Freund's adjuvant, complete Freund's adjuvant, incomplete Freund's adjuvant, CRL-8300 adjuvant, muramyl dipeptide, TLR-4 agonists, flagellin, flagellins derived from gram-negative bacteria, TLR-5 agonists, flagellin fragments capable of binding to TLR-5 receptors, QS-21, ISCOMS, Chitosan, a combination of saponins with sterols and lipids.

62. Immunogenic composition of lyophilized / freeze-dried MMR, according to claim 50, characterized in that it comprises a pharmaceutically acceptable additive selected from a group consisting of a carrier, excipient, binder, isotonic agent, emulsifier and humectant.

63. Immunogenic composition of lyophilized / freeze-dried MMR, according to claim 50, characterized in that the excipient is selected from a group consisting of salt including NaCl, KCl, KH2PO4, Na2HPO4.2H2O, CaCl2 and MgCb; non-ionic surfactant including polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, oxytoxinol 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, and cyclodextrin.

64. Freeze-dried / lyophilized MMR immunogenic composition according to claim 50, characterized in Petition 870250085909, dated 09 / 23 / 2025, pp. 361 / 377 15 / 18 by the fact that the freeze-dried / lyophilized viral vaccine composition is reconstituted with an aqueous solution selected from a group consisting of saline solution, buffer and WFI (water for injection).

65. Immunogenic composition of lyophilized / freeze-dried MMR, according to claim 64, characterized in that the buffer is selected from a group consisting of sodium chloride, acetate, carbonate, citrate, lactate, gluconate, tartrate, phosphate buffer, saline solution, borate, histidine buffer, succinate buffer, HEPES, TRIS and citrate-phosphate.

66. Lyophilized / freeze-dried MMR immunogenic composition according to claim 64, characterized in that the final pH of the reconstituted composition is in the range of pH 6.5 to 7.

5.

67. Immunogenic composition of lyophilized / freeze-dried MMR, according to any one of claims 50 to 66, characterized in that it comprises: a) live attenuated measles virus present in a dose of not less than 1000 CCIDso per dose, live attenuated mumps virus present in a dose of not less than 5000 CCIDso per dose and live attenuated rubella virus present in a dose of not less than 1000 CCIDso per dose; b) stabilizer comprising carbohydrate consisting of sorbitol present in 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 Petition 870250085909, dated 09 / 23 / 2025, p.362 / 377 16 / 18 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. Immunogenic composition of lyophilized / freeze-dried MMR, according to claim 67, characterized in that it comprises: a) live attenuated measles virus present in a dose of not less than 1000 CCIDso per dose, live attenuated mumps virus present in a dose of not less than 5000 CCIDso per dose and live attenuated rubella virus present in a dose of not less than 1000 CCIDso per dose; b) stabilizer comprising carbohydrate consisting of sorbitol present in a concentration of 5% (w / v); amino acid consisting of tricine present in a concentration of 0.3% (w / v), L-histidine present in a concentration of 0.21% (w / v), L-alanine present in a concentration of 0.1% (w / v) and L-arginine hydrochloride present in a concentration of 1.6% (w / v); and hydrolyzed protein consisting of gelatin present at a concentration of 2.5% (w / v) and hydrolyzed lactalbumin present at a concentration of 0.35% (w / v).

69. Lyophilized / freeze-dried MMR immunogenic composition according to claim 68, characterized in that the lyophilized viral vaccine composition is in the form of a single-dose composition or a multiple-dose composition.

70. Lyophilized / freeze-dried MMR immunogenic composition according to claim 69, characterized in that the multi-dose composition additionally comprises a preservative. Petition 870250085909, dated 09 / 23 / 2025, pp. 363 / 377 17 / 18 71. Kit comprising the lyophilized / freeze-dried MMR immunogenic composition, as defined in claim 67 or 68, characterized in that it comprises: a) a first container containing a lyophilized (freeze-dried) viral vaccine composition, said composition comprising: live attenuated measles virus present in a dose of not less than 1000 CCIDso per dose, live attenuated mumps virus present in a dose of not less than 5000 CCIDso per dose and live attenuated rubella virus present in a dose of not less than 1000 CCIDso per dose; carbohydrate consisting of sorbitol present in a concentration of 1 to 10% (w / v); amino acid consisting of tricine present in a concentration of 0.1% to 2% (w / v), L-histidine present in a concentration of 0.1% to 2% (w / v), L-alanine present in a concentration of 0.01% to 1% (w / v) and L-arginine hydrochloride present in a concentration of 0.1% to 5% (w / v);and hydrolyzed protein, consisting of gelatin present in a concentration of 0.1% to 5% (w / v) and hydrolyzed lactalbumin present in a concentration of 0.1% to 2% (w / v); and b) a second container containing an aqueous solution selected from saline solution or water for injection (WFI) for the reconstitution of the lyophilized (freeze-dried) vaccine composition.

72. Kit comprising the lyophilized / freeze-dried MMR immunogenic composition, as defined in claim 67 or 68, characterized in that it comprises: a) a first container containing a lyophilized (freeze-dried) viral vaccine composition, said composition comprising: live attenuated measles virus present in a dose of not less than 1000 CCIDso per dose, live attenuated mumps virus present in a dose of not less than 5000 CCIDso per dose and live attenuated virus. Petition 870250085909, dated 09 / 23 / 2025, page 1.364 / 377 18 / 18 rubella vaccine present in a dose of not less than 1000 CCIDs per dose; 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); and b) a second container containing an aqueous solution selected from saline solution or water for injection (WFI) for reconstitution of the lyophilized (freeze-dried) vaccine composition.

73. Use of the immunogenic composition as defined in any of claims 51 to 70, characterized in that it is for the manufacture of a medicament or vaccine for use in therapy. Petition 870250085909, dated 09 / 23 / 2025, pp. 365 / 377

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