Method of producing immunogenic composition

Abstract

To provide, in particular, a method of producing an immunogenic composition exhibiting reduced virucidal activity, as well as the immunogenic composition and uses thereof.SOLUTION: The method in particular comprises the steps of: (a) providing a mixture with a first liquid and a recombinant protein, (b) concentrating the recombinant protein in the mixture by removing a portion of the first liquid from the mixture, and (c) processing the solution resulting from step (b) by continuous diafiltration.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to methods for producing immunogenic compositions and to such immunogenic compositions which exhibit reduced virucidal activity. [Background technology]

[0002] Porcine circovirus type 2 (PCV2) is a small (17-22 nm diameter), icosahedral, non-enveloped DNA virus that contains a single-stranded circular genome. PCV2 shares approximately 80% sequence identity with porcine circovirus type 1 (PCV1). However, in contrast to PCV1, which is generally non-pathogenic, pigs infected with PCV2 usually exhibit a syndrome called postweaning multisystemic wasting syndrome (PMWS). PMWS is characterized clinically by emaciation, pale skin, failure to thrive, respiratory distress, diarrhea, icterus, and jaundice. In some affected pigs, a combination of all symptoms is evident, whereas others have only one or two of these symptoms. At necropsy, microscopic and gross lesions are also evident in multiple tissues and organs, with lymphoid tissue being the most common site of lesion. A strong correlation has been observed between the amount of PCV2 nucleic acid or antigen and the severity of microscopic lymphoid lesions. Mortality rates for pigs infected with PCV2 can reach 80%. In addition to PMWS, PCV2 is associated with several other infectious diseases, including pseudorabies, porcine reproductive and respiratory syndrome (PRRS), Glasser's disease, streptococcal meningitis, salmonellosis, postweaning colibacillosis, dietary hepatopathy, and suppurative bronchopneumonia.

[0003] Several vaccines are available to reduce the impact of PCV2 infection in pigs. US Patent 6,703,023 provides a DNA-based vaccine for the prevention of pigs against PMWS. In WO 03 / 049703, the production of a live chimeric vaccine is described, which contains the non-pathogenic PCV1 virus, but the ORF2 protein is replaced by the ORF2 protein of the pathogenic PCV2. WO 99 / 18214 and WO 99 / 29717 provide procedures for the preparation of several PCV2 strains and PCV2 killed vaccines. The preparation of subunit vaccines is also described in WO 99 / 18214 and WO 99 / 29717. An effective ORF2-based subunit vaccine is reported in WO 06 / 072065. Further ORF2-based subunit vaccines are also described in WO 07 / 28823.

[0004] The open reading frame 2 (ORF2) protein of PCV2, which has a molecular weight of approximately 30 kDa when run on SDS-PAGE gel, has been utilized as an antigen component in vaccines and immunogenic compositions for PCV2 so far.The typical method of obtaining ORF2 for use in such vaccines and compositions generally consists of amplifying the PCV2 DNA encoding ORF2, expressing ORF2 protein in host cells, and extracting ORF2 protein from host cells by cell lysis.The ORF2 cell lysate recovered is then used as the antigen portion of immunogenic compositions or vaccines.In some cases, the cell lysate containing ORF2 is separated from cell debris. Immunogenic compositions against PCV2 and various immunogenic compositions against other pathogens often have virucidal effect against other antigens.For example, current regulatory standards in the United States (9 CFR 113.35) allow some virucidal activity in multivalent compositions, but this virucidal activity must not cause more than 0.7 log / mL loss of live virus or less than 0.7 log / mL CFU loss of live bacteria when combined with other components of immunogenic compositions.Compositions with unacceptable virucidal activity cannot be combined with other antigens to create multivalent vaccines. Summary of the Invention

[0005] To this end, WO 11 / 28888 provides a method for reducing the virucidal activity of a composition comprising a PCV2 antigen, as well as antigenic preparations and immunogenic compositions comprising a PCV2 antigen, which have reduced virucidal activity. More particularly, WO 11 / 28888 provides a method for producing such a PCV2 antigenic composition on a large scale, the method comprising: i) obtaining a first liquid containing a PCV2 antigen comprising virus-like particles of ORF2 protein; and ii) removing at least a portion of the first liquid from the PCV2 antigen, including virus-like particles of ORF2 protein, by a filtration step using a filter, the filter comprising a semipermeable membrane having an average pore size smaller than the PCV2 antigen, thereby preventing at least 90% of the PCV2 antigen from passing through the pores of the semipermeable membrane and retaining the PCV2 antigen within the filter; Including, A portion of the first liquid is removed from the PCV2 antigen by exchange of a portion of the first liquid for a second liquid, the second liquid being different from the first liquid and The exchange of a portion of the first liquid with a second liquid a) a step of adding a liquid comprising adding a second liquid to a first liquid containing a PCV2 antigen; and b) concentrating the PCV2 antigen by 3-50 times compared to the volume of the first liquid by removing a portion of the first and second liquids. The present invention teaches a method including:

[0006] However, said liquid addition step a), which involves adding the second liquid, preferably in excess volume relative to the first liquid, may result in limitations with respect to the total production scale volume, since the resulting volumes of the first and second liquids are usually increased by a factor compared to the first liquid, prior to the concentration step b). Therefore, additional methods for producing immunogenic compositions containing recombinant proteins with reduced virucidal effects on larger and even very large scales are desirable. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] The solution of the above technical problem is achieved by the description and embodiments characterized in the claims. The invention in its different aspects is therefore carried out according to the appended claims. The present invention is based on the surprising finding that a method for producing an immunogenic composition comprising a recombinant protein is sufficient for the desired purpose, wherein the method comprises, preferably in the following order: (a) - a first liquid, and - a recombinant protein and / or a quaternary structure comprising a plurality of said recombinant proteins providing a mixture containing (b) concentrating the recombinant protein and / or said quaternary structure in the mixture resulting from step (a) by removing a portion of the first liquid from the mixture; and (c) treating the solution resulting from step (b) by continuous diafiltration, in particular by continuous diafiltration against a second liquid, the second liquid being different from the first liquid, or these steps Includes. As used herein, the term "first liquid" is understood to be equivalent to the terms "initial liquid" or "liquid(1)", respectively. Thus, the term "first liquid" is interchangeable with the term "liquid(1)" or the term "initial liquid". It is further understood that as used herein, the term "second liquid" is equivalent to the terms "additional liquid" or "liquid (2)", respectively. Thus, the term "second liquid" is interchangeable with the term "liquid (2)" or the term "additional liquid".

[0008] In one preferred embodiment, the mixture of step (a) additionally contains a vector comprising a nucleic acid sequence encoding said recombinant protein, said vector being inactivated by an inactivating agent. According to another preferred embodiment, the mixture of step (a) additionally contains an inactivating agent that has been neutralized by a neutralizing agent. In yet another preferred embodiment, the mixture of step (a) additionally contains a neutralizing agent. As referred to herein, it is understood in particular that the term "mixture of step (a)" is synonymous with "mixture provided in step (a)." As used herein, it is understood in particular that the term "provided in a step" is equivalent to "provided by a step." The present invention relates to a method as described and / or claimed herein, wherein the mixture of step (a) above comprises: - a vector comprising a nucleic acid sequence encoding said recombinant protein, said vector being inactivated by an inactivating agent, and / or - the inactivating agent has been neutralized by a neutralizing agent, and / or - Neutralizer and Preferably, the mixture of step (a) additionally comprises said vector, said neutralized inactivating agent and a neutralizing agent.

[0009] Furthermore, in step (c), the solution resulting from step (b) is preferably treated by continuous diafiltration such that the concentration of neutralized inactivating agent and / or the concentration of neutralizing agent is reduced in the treated solution. Before describing the aspects of the present invention, it should be noted that, as used herein and in the appended claims, the singular forms "a", "an" and "the" include the corresponding plural forms unless the context clearly indicates otherwise. Thus, for example, a reference to "an antigen" includes a plurality of antigens, a reference to "virus" is a reference to one or more viruses and their equivalents known to those skilled in the art, and so forth. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices and materials are described herein. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing the cell lines, vectors and methodologies reported in the publications that may be used in connection with the present invention. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by prior invention.

[0010] In one embodiment, the present invention provides a method for producing an immunogenic composition comprising a recombinant protein, preferably comprising the steps of: (a)(i) - a first liquid; - a recombinant protein and / or a quaternary structure comprising a plurality of said recombinant proteins, - a vector comprising a nucleic acid sequence encoding said recombinant protein providing a mixture containing (ii) inactivating the vector by adding an inactivating agent to the mixture of step (i); (iii) neutralizing the deactivating agent by adding a neutralizing agent to the mixture resulting from step (ii); (b) concentrating the recombinant protein and / or said quaternary structure in the mixture resulting from step (a)(iii) by removing a portion of the first liquid from the mixture; and (c) treating the solution resulting from step (b) by continuous diafiltration such that the concentration of neutralized inactivating agent and / or the concentration of neutralizing agent is reduced in the treated solution. The present invention relates to a method comprising or including the steps of:

[0011] In particular, the continuous diafiltration of step (c) is continuous diafiltration against a second liquid, the second liquid being different from the first liquid. In the context of the present invention, it is understood in particular that "(i)", "(ii)" and "(iii)" represent sub-steps of step (a) or that step (a) consists of or includes the sub-steps "(i)", "(ii)" and "(iii)", respectively, and thus (i) is equivalent to "(a)(i)", (ii) is equivalent to "(a)(ii)", and (iii) is equivalent to "(a)(iii)". In particular, when referred to herein, the terms "mixture of step (i)" and "mixture of step (a)(i)" are understood to be synonymous with "mixture provided in step (i)" and "mixture provided in step (a)(i)", respectively.

[0012] For the purposes of the present invention, a "first liquid" refers to a liquid, aqueous or flowable medium that is typically used in combination with cells, antigens, immunogenic compositions, vaccines, etc. Preferably, the first liquid comprises a medium from an antigen composition, more preferably, the first liquid comprises or preferably consists of a cell culture medium used for the production of recombinant proteins in cultured host cells. The cultured host cells may be bacteria, yeast, insect cells, animal cells and mammalian cells, with insect and mammalian cells being particularly preferred. Thus, the first liquid may comprise or consist of a medium for the culture of bacteria, yeast, insect cells, animal cells or mammalian cells. Preferably, the cell culture medium is a serum-free cell culture medium, most preferably, the culture medium is Excell 420 serum-free medium when insect cells are used.

[0013] In another aspect, the present invention therefore relates to the methods as described and claimed herein, wherein said first liquid comprises part of or consists of a cell culture medium, which is preferably an insect cell culture medium. For purposes of the present invention, "portion" refers to any amount that does not include the entire amount. For example, a portion of a liquid is an amount that is less than 100% of the volume of the liquid, such as 90% of the liquid, 80% of the liquid, 70% of the liquid, and any amount greater than 0% but less than 100%. As used herein, the term "recombinant protein" refers specifically to a protein produced by recombinant DNA techniques, generally by inserting DNA encoding the expressed protein into a suitable expression vector, which is then used to transform or, in the case of a viral vector, infect a host cell to produce the heterologous protein. Thus, as used herein, the term "recombinant protein" refers specifically to a protein molecule expressed from a recombinant DNA molecule. As used herein, a "recombinant DNA molecule" refers to a DNA molecule that is composed of segments of DNA linked together by molecular biological techniques. Suitable systems for the production of recombinant proteins include, but are not limited to, insect cells (e.g., baculovirus), prokaryotic systems (e.g., E. coli), yeast (e.g., Saccaromyces cerevisiae, Pichia pastoris), mammalian cells (e.g., Chinese hamster ovary, HEK293), plants (e.g., safflower), avian cells, amphibian cells, fish cells, and cell-free systems (e.g., rabbit reticulocyte lysate).

[0014] In the context of the present invention, the recombinant protein is preferably a recombinant PCV2 ORF2 protein. The term "quaternary structure" as used in the context of the present invention particularly relates to the assembly of multiple folded protein molecules in multi-subunit complexes, and in particular to virus-like particles. For the purposes of the present invention, a "quaternary structure comprising a plurality of said recombinant proteins" refers to a three-dimensional arrangement of a plurality of said recombinant proteins, such as a virus-like particle comprising a plurality of said recombinant proteins. In this regard, it is understood that the term "a plurality of said recombinant proteins" is equivalent to "a plurality of recombinant proteins". In particular, it is understood that said term refers to a virus-like particle comprising several molecules of a particular recombinant protein, for example, a recombinant PCV2 ORF2 protein. In the context of the present invention, "inactivating a vector" means, in particular, rendering a vector that is normally replication-competent unable to replicate. "Inactivation" refers, in particular, to the process of inactivating a vector.

[0015] For the purposes of the present invention, "inactivating agent" refers to any agent that can be used in any conventional inactivation method. Inactivation can be performed by chemical and / or physical treatments known to those skilled in the art. Preferred inactivating agents include cyclized binary ethyleneimine (BEI), including a solution of 2-bromoethyleneamine hydrobromide (BEA), cyclized to binary ethyleneimine (BEI). Further preferred chemical inactivating agents include, but are not limited to, Triton X-100, sodium deoxycholate, cetyltrimethylammonium bromide, β-propiolactone, thimerosal, phenol, and formaldehyde (formalin). Thus, as used herein, the term "inactivating agent" particularly relates to a chemical agent capable of modifying a vector by a chemical reaction such that the vector is unable to replicate. Preferably, the inactivating agent is an aziridine compound, in particular binary ethyleneimine (BEI), and / or the inactivating agent is added in molar excess relative to the vector, in particular in molar excess relative to the nitrogen bases of the vector DNA base pairs that react with BEI.

[0016] For the purposes of the present invention, "neutralizing agent" refers to any agent capable of neutralizing the inactivating agent described herein, such that the inactivating agent is no longer capable of inactivating vectors. When an aziridine compound is used for inactivation, a nucleophilic agent that opens the three-membered ring is preferably used for neutralization. The agent that neutralizes the inactivating agent is preferably sodium thiosulfate, sodium bisulfite, etc. In particular, it is preferred that the neutralizing agent is sodium thiosulfate and / or that the neutralizing agent is added in molar excess relative to the inactivating agent. As described herein, "neutralized inactivating agent" particularly refers to the product or products resulting from the chemical reaction of a inactivating agent with a neutralizing agent, e.g., the products of the chemical reaction of BEI with thiosulfate or other nucleophiles. Any conventional chemical inactivation method can be used to inactivate the vector. In a preferred form, for chemical treatment, the temperature is between about 32°C and 42°C, more preferably between about 34°C and 40°C, and most preferably between about 35°C and 39°C. A preferred inactivation method includes the addition of cyclized binary ethyleneimine (BEI) at a concentration of preferably about 1 to about 20 mM, preferably about 2 to about 10 mM, even more preferably about 2 to about 8 mM, even more preferably about 3 to about 7 mM, and most preferably about 5 mM. For example, inactivation preferably includes the addition of a solution of about 0.4 M 2-bromoethyleneamine hydrobromide (BEA), which is cyclized to 0.2 M binary ethyleneimine (BEI) in 0.3 N NaOH to give the fluid a final concentration of about 5 mM BEI. Preferably, the fluid is then continuously stirred for 2 to 96 hours. After inactivation is complete, sodium thiosulfate solution, preferably 1.0 M sodium thiosulfate solution, is added to neutralize any remaining BEI, and the inactivated / neutralized collection fluid may be stored frozen at -40°C or below, or between about 1°C and 7°C. Preferably, sodium thiosulfate is added in an equivalent amount compared to the BEI previously added for inactivation. For example, in the case where BEI is added at a final concentration of 5 mM, 1.0 M sodium thiosulfate solution is added to give a final minimum concentration of 5 mM to neutralize any remaining BEI.

[0017] Thus, in step (iii), the neutralizing agent is preferably added in an equivalent amount compared to the amount of deactivating agent added in step (ii). In the context of ranges, when described herein, it is understood that "y to z" is equivalent to "y to z", and both are equivalent to "y to z", respectively, in particular, for example, "0.1 to 1.0" is equivalent to "0.1 to 1.0".

[0018] For purposes of the present invention, "vector" and "vector comprising a nucleic acid sequence encoding said recombinant protein" refer to an appropriate expression vector, preferably a baculovirus expression vector, which is then used to transfect, or in the case of a baculovirus expression vector, infect a host cell so as to produce the protein or polypeptide encoded by the DNA. Vectors and methods for making and / or using vectors (or recombinants) for expression are described in U.S. Pat. Nos. 4,603,112, 4,769,330, 5,174,993, 5,505,941, 5,338,683, 5,494,807, 4,722,848, 5,942,235, 5,364,773, 5,762,938, 5,770,212, 5,942,235, 382,425, PCT publications WO 94 / 16716, WO 96 / 39491, WO 95 / 30018; Paoletti, "Applications of pox virus vectors to vaccination: An update," PNAS USA 93: 11349-11353, October 1996;Moss, "Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety," PNAS USA 93: 11341-11348, October 1996;Smith et al., U.S. Pat. No. 4,745,051 (recombinant baculovirus);Richardson, CD (Editor), Methods in Molecular Biology 39, "Baculovirus Expression Protocols" (1995 Humana Press Inc.);Smith et al., "Production of Human Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector", Molecular and Cellular Biology, December, 1983, Vol. 3, No. 12, p. 2156-2165;Pennock et al., "Strong and Regulated Expression of Escherichia coli B-Galactosidase in Infect Cells with a Baculovirus vector," Molecular and Cellular Biology, March 1984, Vol. 4, No. 3, p. 406;European Patent Application No. 0370573;U.S. Application No. 920,197, filed October 16, 1986;European Patent Application Publication No. 265785;U.S. Patent No. 4,769,331 (recombinant herpes viruses);Roizman, "The function of herpes simplex virus genes: A primer for genetic engineering of novel vectors," PNAS USA 93:11307-11312, October 1996; Andreansky et al., "The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors," PNAS USA 93: 11313-11318, October 1996; Robertson et al., "Epstein-Barr virus vectors for gene delivery to B lymphocytes", PNAS USA 93: 11334-11340, October 1996; Frolov et al., "Alphavirus-based expression vectors: Strategies and applications," PNAS USA 93: 11371-11377, October 1996; Kitson et al., J. Virol. 65, 3068-3075, 1991; U.S. Patent No. 5,591,439, U.S. Patent No. 5,552,143; International Publication No. WO 98 / 00166; U.S. Application No. 08 / 675,556 and U.S. Application No. 08 / 675,566 (recombinant adenovirus), both filed and allowed on July 3, 1996; Grunhaus et al., 1992, "Adenovirus as cloning vectors," Seminars in Virology (Vol. 3) p. 237-52, 1993;;Ballay et al. EMBO Journal, vol. 4, p. 3861-65, Graham, Tibtech 8, 85-87, April, 1990; Prevec et al., J. Gen Virol. 70, 42434; PCT Publication No. WO 91 / 11525; Felgner et al. (1994), J. Biol. Chem. 269, 2550-2561, Science, 259: 1745-49, 1993; and McClements et al., “Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease”, PNAS USA 93: 11414-11420, October 1996; and U.S. Pat. Nos. 5,591,639, 5,589,466 and 5,580,859, as well as WO 90 / 11092, WO 93 / 19183, WO 94 / 21797, WO 95 / 11307, WO 95 / 20660; particularly with respect to DNA expression vectors, Tang et al., Nature, and Furth et al., Analytical Biochemistry. See also WO 98 / 33510; Ju et al., Diabetologia, 41: 736-739, 1998 (lentiviral expression systems); Sanford et al., U.S. Pat. No. 4,945,050; Fischbach et al. (IntraCell); WO 90 / 01543; Robinson et al., Seminars in Immunology vol. 9, pp. 271-283 (1997) (DNA vector systems); Szoka et al., U.S. Pat. No. 4,394,448 (methods for inserting DNA into living cells); McCormick et al., U.S. Pat. No. 5,677,178 (use of cytopathic viruses); and U.S. Pat. No. 5,928,913 (vectors for gene delivery), as well as other references cited herein.

[0019] Preferred viral vectors include baculoviruses, such as BaculoGold (BD Biosciences Pharmingen, San Diego, Calif.), particularly if the production cells are insect cells. Although the baculovirus expression system is preferred, it will be understood by those of skill in the art that other expression systems, including those described above, will work for the purposes of the present invention, i.e., for expression of recombinant proteins.

[0020] Thus, in the context of the present invention, a "vector" is preferably a recombinant virus, preferably a baculovirus, and / or a "nucleic acid sequence" is preferably a DNA sequence. Preferably, in step (b) of the methods described and claimed herein, the step of removing a portion of the first liquid from the mixture consists of or comprises filtering the mixture through at least one filter, said at least one filter preferably comprising a filter membrane.

[0021] According to another preferred embodiment, in step (b), the concentrating step comprises: - feeding the mixture into a filter system containing at least one filter, the at least one filter comprising a filter membrane having a membrane pore size that allows the passage of the neutralized inactivating agent and / or neutralizing agent while retaining the recombinant protein and / or said quaternary structure in the bulk stream; and - Discharging the permeate containing the neutralized inactivating agent and / or neutralizing agent; Includes.

[0022] According to a further preferred embodiment, in step (c), the continuous diafiltration comprises: - feeding the solution into a filter system containing at least one filter, the at least one filter comprising a filter membrane having a membrane pore size that allows the passage of the neutralized inactivating agent and / or neutralizing agent while retaining the recombinant protein and / or said quaternary structure in the bulk stream; - discharging the permeate containing the neutralized inactivating agent and / or neutralizing agent; and - adding a second liquid to the bulk stream at a rate equal to the permeate stream, the second liquid being different from the first liquid; Includes.

[0023] For the purposes of the present invention, a "second liquid" refers to any liquid that is different from the first liquid and that is normally used in combination with cells, antigens, immunogenic compositions, vaccines, etc. Preferably, the second liquid is an aqueous solution, even more preferably a pharma- ceutical acceptable solution, even more preferably a buffer, most preferably a physiologically acceptable buffer, such as saline or phosphate buffer, e.g. P-saline or PBS. Most preferably, the second liquid is characterized by being not virucidal towards any live virus or live bacteria when the live virus or live bacteria are cultured or stored in such liquid. Preferably, the at least one filter described herein is at least one flat sheet filter and / or at least one hollow fiber filter, and said at least one filter is most preferably at least one flat sheet filter. The at least one flat sheet filter is preferably at least one cassette filter.

[0024] In particular, the at least one filter is 2 to 8 filters, preferably 5 to 7 filters, most preferably 6 filters, and / or each of the at least one filter is, in particular, about 16 to 26 m 2 , preferably about 20 to 22 m 2 , most preferably about 21 m 2has a total filter area of As described herein, the filter membrane preferably has an average pore size smaller than the recombinant protein and / or an average pore size smaller than said quaternary structure, and / or the filter membrane preferably has a molecular weight cut-off of between about 200 kDa and about 500 kDa, in particular about 300 kDa.

[0025] Preferably, the filter membrane is made of or comprises a material selected from the group consisting of polyethersulfone, cellulose hydrate, regenerated cellulose, stabilized cellulose, crosslinked cellulose, crosslinked cellulose hydrate, cellulose acetate, polyamide, polyurethane, polypropylene, polysulfone, polycarbonate, nylon, polyimide and combinations thereof; And / or the filter membrane preferably consists of or comprises polyethersulfone, or the filter membrane may be a stabilized cellulosic membrane. According to another preferred embodiment of the present invention, in step (b) and in step (c), the same filter system is utilized. Thus, according to this preferred embodiment, a single filter system is used for both the concentration in step (b) and the continuous diafiltration in step (c). Preferably, the mixture remaining after step (c) contains a concentration of deactivating agent that is less than 1000 times the concentration of the neutralising agent resulting from step (iii).

[0026] Furthermore, in the context of the present invention: - step (iii) is carried out in a first container, the mixture resulting from the step of neutralizing the deactivating agent is transferred from the first container to a second container connected to a filter system, after transferring the mixture from the first container to the second container, the valve between the first container and the second container is closed and the empty first container is filled with the second liquid, - in step (b), the mixture is circulated through the second vessel and the filter system until concentration is complete; and - in step (c), while the mixture is circulating through the filter system and the second container, a valve between the first and second containers is opened and a second liquid is continuously introduced from the first container to the second container; It has been found that this is particularly preferable. In a further aspect, the present invention relates to the methods and claimed methods described herein, wherein the volume of the mixture resulting from step (a) is between 1000L and 10000L, preferably between 2000L and 8000L, most preferably between 3000L and 5000L.

[0027] When referred to herein, it is understood that the "volume of the mixture resulting from step (a)" is in particular equivalent to the "initial volume of the mixture in step (b) prior to said concentration." In another aspect, the invention relates to a method as described and claimed herein, wherein in step (b) the recombinant protein and / or the quaternary structure is finally concentrated at least 5-fold, preferably at least 8-fold, more preferably 9-fold to 11-fold, most preferably about 10-fold, compared to the volume of the mixture resulting from step (a). The concentrating step (b) of the method provided herein may be performed such that the recombinant protein or said quaternary structure is concentrated 3-fold to 50-fold compared to the volume of the mixture obtained from step (a) or the volume of the mixture obtained from step (iii), respectively. More preferably, the concentrating step may be performed such that the recombinant protein or said quaternary structure is concentrated at least 5-fold, such as 5-fold to 20-fold, compared to the volume of the mixture obtained from step (a) or the volume of the mixture obtained from step (iii), respectively. Even more preferably, the concentrating step may be performed such that the recombinant protein or said quaternary structure is concentrated at least 8-fold, such as 8-fold to 14-fold, compared to the volume of the mixture obtained from step (a) or the volume of the mixture obtained from step (iii), respectively. Most preferably, the concentrating step is performed such that the recombinant protein or said quaternary structure is concentrated at least 10-fold, such as 10-fold to 12-fold, compared to the volume of the mixture obtained from step (a) or the volume of the mixture obtained from step (iii), respectively.

[0028] Preferably, in step (c), the total volume of the second liquid added to the bulk stream is at least 5 times, preferably at least 7 times, more preferably at least 9 times, most preferably at least 10 times the volume of the solution resulting from step (b), and / or the total volume of the second liquid added to the bulk stream is most preferably about the same as the volume of the mixture resulting from step (a). In a further aspect, the present invention provides a method as described and claimed herein, comprising: (d) mixing the mixture remaining after step (c) with a further component selected from the group consisting of pharma- ceutically acceptable carriers, adjuvants, diluents, excipients and combinations thereof, wherein the concentration of recombinant protein and / or the concentration of said quaternary structure in the solution resulting from said mixing step is preferably about the same or lower (e.g., 0.2-0.5 times) than the concentration of recombinant protein and / or said quaternary structure in the mixture resulting from step (a). The present invention relates to a method comprising the steps of:

[0029] When referred to in this specification, the "concentration of recombinant protein and / or said quaternary structure in the mixture resulting from step (a)" is understood to be equivalent in particular to the "initial concentration of recombinant protein and / or said quaternary structure in the mixture in step (b), prior to said concentrating step". As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, absorption delaying agents and the like.

[0030] "Adjuvants" as used herein can include aluminum hydroxide and aluminum phosphate, saponins such as Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), water-in-oil emulsions, oil-in-water emulsions, and water-in-oil-in-water emulsions. The emulsions can be based on light liquid paraffin oil (European Pharmacopoeia type); isoprenoid oils such as squalane or squalene; oils obtained from the oligomerization of alkenes, especially isobutene or decene; esters of acids or alcohols containing linear alkyl groups, more particularly vegetable oils, ethyl oleate, propylene glycol di(caprylate / caprate), glyceryl tri(caprylate / caprate), or propylene glycol dioleate; esters of branched fatty acids or alcohols, especially isostearate esters. The oils are used in combination with emulsifiers to form the emulsions. The emulsifier is preferably a non-ionic surfactant, in particular sorbitan, mannides (e.g., anhydrous mannitol oleate), glycols, polyglycerols, propylene glycol, and esters of oleic acid, isostearic acid, linoleic acid or hydroxystearic acid, which may be ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular Pluronic products, in particular Pluronic L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed.Stewart-Tull, DES). JohnWiley and Sons, NY, pp 51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997).For example, it is possible to use the SPT emulsion described on page 147 of "Vaccine Design, The Subunit and Adjuvant Approach" edited by M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of the same book. Further suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), block copolymers (CytRx, Atlanta Ga), SAF-M (Chiron, Emeryville Calif.), monophosphorys lipid A, avridine lipid-amine adjuvant, heat-labile enterotoxin from Escherichia coli (recombinant or otherwise), cholera toxin, IMS1314 or muramyl dipeptide, among many others. Among the copolymers of maleic anhydride and alkenyl derivatives is the copolymer EMA (Monsanto), which is a copolymer of maleic anhydride and ethylene. Dissolution of these polymers in water results in an acid solution that is preferably neutralized to physiological pH to provide an adjuvant solution that is incorporated into the immunogenic, immunological or vaccine composition itself.

[0031] Further examples of adjuvants are compounds selected from polymers of acrylic or methacrylic acid and copolymers of maleic anhydride and alkenyl derivatives. Advantageous adjuvant compounds are in particular polymers of acrylic or methacrylic acid crosslinked with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). The skilled artisan may also refer to US Pat. No. 2,909,462, which describes such acrylic polymers crosslinked with polyhydroxylated compounds having at least three hydroxyl groups, preferably not more than eight, in which at least three hydroxyl hydrogen atoms are replaced by unsaturated aliphatic radicals having at least two carbon atoms. Preferred radicals are those containing 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. Particularly suitable are the products sold under the name Carbopol (BF Goodrich, Ohio, USA). They are crosslinked with allyl sucrose or allyl pentaerythritol. Among these, Carbopol 974P, 934P and 971P may be mentioned. Most preferably, Carbopol 971P is used.

[0032] "Diluents" can include water, saline, dextrose, ethanol, glycerol, and the like. "Isotonic agents" can include, inter alia, sodium chloride, dextrose, mannitol, sorbitol, and lactose. "Stabilizers" include, inter alia, albumin and alkali salts of ethylenediaminetetraacetic acid. "Preservatives" as used herein refer to antimicrobial active agents such as, for example, gentamicin, merthiolate, etc. In particular, the addition of preservatives is most preferred for the preparation of multi-dose compositions. These antimicrobial active agents are added at a concentration effective to prevent the subject composition from any microbial contamination or to inhibit any microbial growth within the subject composition.

[0033] Preferably, in step (d), said further component is a combination of an adjuvant, water and sodium chloride, in particular a combination of an adjuvant solution and P-saline, said adjuvant being preferably a polymer of acrylic or methacrylic acid, even more preferably a carbomer. As referred to herein, the term "P-saline" particularly relates to a buffer solution composed of 0.8-0.9% (w / v) NaCl, e.g., 0.85% (w / v) NaCl, dissolved in water and adjusted to a pH of 6.8-7.0.

[0034] In another aspect, the present invention relates to the methods and claimed methods described herein, wherein the mixture used for step (a) comprises: (1) allowing infection of susceptible cells in culture with a vector comprising a nucleic acid sequence encoding said recombinant protein, said recombinant protein being expressed by said vector; (2) subsequently recovering the recombinant protein and / or quaternary structures comprising said recombinant proteins from the cell culture, preferably in which the cell debris is separated from the recombinant protein and / or said quaternary structures by a separation step, preferably comprising microfiltration through at least one filter, preferably two filters, the at least one filter preferably having a pore size larger than the recombinant protein and / or the quaternary structures comprising said recombinant proteins, in particular having a pore size of about 1 to about 20 μm and / or about 0.1 μm to about 4 μm. The present invention relates to a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: Preferably, in the method, the separation step comprises: - microfiltration through one or more filters having a pore size of about 2 μm to about 15 μm, and / or - comprising or consisting of microfiltration through one or more filters having a pore size of about 0.8 μm to about 1.0 μm.

[0035] Preferred cells are cells susceptible to infection with an appropriate recombinant viral vector that contains the DNA for the recombinant protein and expresses the recombinant protein. Preferably, the cells are insect cells, more preferably these include insect cells sold under the trademark SF+ insect cells (Protein Sciences Corporation, Meriden, CT). A preferred cell culture is a culture of approximately 0.3-2.0 x 10 6 cells / mL, more preferably about 0.35 to 1.9 × 10 6 cells / mL, and even more preferably about 0.4-1.8×10 6 cells / mL, and even more preferably about 0.45-1.7×10 6 cells / mL, most preferably about 0.5-1.5×10 6 With a cell count between 100 and 150 cells / mL. The vectors containing the nucleic acid sequence encoding the recombinant protein, when used for infection of susceptible cells, have a preferred multiplicity of infection (MOI) of between about 0.03-1.5, more preferably about 0.05-1.3, even more preferably about 0.09-1.1, and most preferably about 0.1-1.0. Preferably, the above mentioned MOI is for 1 mL of cell culture fluid. Preferably, the method described herein comprises the step of infecting a susceptible cell with a recombinant protein DNA and a recombinant viral vector expressing the recombinant protein with an MOI (multiplicity of infection) of between about 0.03-1.5, more preferably about 0.05-1.3, even more preferably about 0.09-1.1, and most preferably about 0.1-1.0, of 0.35-1.9×10 6 cells / mL, and even more preferably about 0.4-1.8×10 6cells / mL, and more preferably, about 0.45 to 1.7 x 10 6 cells / mL, most preferably about 0.5-1.5×10 6 Including infection in cells / mL.

[0036] Appropriate growth media can also be determined by one of skill in the art, with a preferred growth medium being a serum-free insect cell medium such as Excell 420 (JRH Biosciences, Inc., Lenexa, Kans.). In said method, it is preferred that the cell culture in step (1) is preferably maintained at 22-32°C, more preferably about 24-30°C, also more preferably about 25-29°C, even more preferably 26-28°C, most preferably 27°C, while the recombinant protein is expressed by the vector, and / or that the harvesting in step (2) occurs 5-8 days, most preferably day 8, after inoculation of the cells with the vector and / or when cell viability has decreased to less than 10%.

[0037] In another aspect, the present invention relates to the methods and claimed methods described herein, wherein the recombinant protein comprises: - a recombinant PCV2 ORF2 protein, which preferably comprises or alternatively consists of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.5% or 100% sequence identity with the sequence of SEQ ID NO:1 or SEQ ID NO:2. The present invention relates to a method comprising the steps of: As used herein, the term "having 100% sequence identity" is understood to be equivalent to the term "identical." Percent sequence identity has an art-recognized meaning, and there are many methods for measuring the identity between two polypeptide or polynucleotide sequences.See, for example, Lesk, Ed., Computational Molecular Biology, Oxford University Press, New York, (1988);Smith, Ed., Biocomputing: Informatics And Genome Projects, Academic Press, New York, (1993);Griffin & Griffin, Eds., Computer Analysis Of Sequence Data, Part I, Humana Press, New Jersey, (1994);von Heinje, Sequence Analysis In Molecular Biology, Academic Press, (1987);and Gribskov & Devereux, Eds., Sequence Analysis Primer, M Stockton Press, New York, (1991). Methods for aligning polynucleotides or polypeptides have been codified in computer programs and include those in the GCC program package (Devereux et al., Nuc. Acids Res. 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al., J. Molec. Biol. 215:403 (1990)), and the Bestfit program (Wisconsin Sequence Analysis Package, version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) which uses the local homology algorithm of Smith and Waterman (Adv. App. Math., 2:482-489 (1981)).For example, the computer program ALIGN can be used, which utilizes the FASTA algorithm, using an affine gap search with a gap opening penalty of -12 and a gap extension penalty of -2. For purposes of the present invention, protein / amino acid sequences are aligned using the Clustal W method in MegAlign software version 11.1.0(59),419 from DNASTAR Inc., using the default multiple alignment parameters set in this program (Gonnet's set of protein weights matrix with gap penalty=10.0, gap length penalty=0.2, and delayed mismatched sequences (%)=30%).

[0038] As used herein, in particular, the term "sequence identity to the sequence of SEQ ID NO:X" is understood to be equivalent to the terms "sequence identity to the sequence of SEQ ID NO:X over the entire length of SEQ ID NO:X" or "sequence identity to the sequence of SEQ ID NO:X over the entire length of SEQ ID NO:X", respectively. In this context, "X" is any integer selected from 1 to 2, such that "SEQ ID NO:X" represents any of the SEQ ID NOs referred to herein.

[0039] It is further understood that the term "recombinant protein consisting of a sequence" also relates to any co-translational and / or post-translational modification or modifications of the sequence, in particular those modifications or modifications that are brought about by the cell expressing the polypeptide. Thus, as described herein, the term "recombinant protein consisting of a sequence" also covers sequences having one or more modifications brought about by the cell expressing the polypeptide, in particular those modifications of amino acid residues brought about in protein biosynthesis and / or protein processing, preferably selected from the group consisting of glycosylation, phosphorylation and acetylation. In another aspect, the present invention relates to the methods and claimed methods described herein, wherein said quaternary structure comprising a plurality of said recombinant proteins is a virus-like particle comprising a plurality of recombinant PCV2 ORF2 proteins.

[0040] In another aspect, the present invention relates to the methods and claimed methods described herein, wherein the virucidal activity of the mixture resulting from said method is reduced by at least 20% compared to a mixture that has not been subjected to the concentration of step (b) and the continuous diafiltration of step (c) of said method, and / or the immunogenic composition produced by said method has a virucidal activity of at least 1 log TCID 3 per mL of live virus when the live virus is mixed with the immunogenic composition for 4 hours or more, in particular at room temperature. 50 As referred to herein, "room temperature" particularly relates to a temperature between 20 and 25 degrees Celsius, more particularly 23°C (on average). In another aspect, the invention relates to the methods and claimed methods described herein, wherein the live virus is Porcine Reproductive and Respiratory Syndrome (PRRS) virus.

[0041] In another aspect, the present invention relates to a method as described and claimed herein, further comprising the step of mixing the mixture remaining after step (c) and / or step (d) with at least one additional antigen, wherein the at least one additional antigen is preferably Porcine Reproductive and Respiratory Syndrome (PRRS) virus. In the context of the present invention, it is understood in particular that the term "remaining after the step" is equivalent to "obtained from the step". The present application not only provides methods for producing immunogenic compositions, but also relates to immunogenic compositions produced by the methods described and claimed herein. In another aspect, the present invention therefore relates to an immunogenic composition obtainable by the methods described and claimed herein. According to a specific preferred embodiment, the immunogenic composition is a solution containing 1 to 10 μM L-arginine and / or 1 to 10 μM L-lysine, and most preferably, the immunogenic composition is a solution containing 1 to 10 μM L-arginine and 1 to 10 μM L-lysine.

[0042] Thus, the present invention provides - a recombinant PCV2 ORF2 protein, which preferably consists of or comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.5% or 100% sequence identity with the sequences SEQ ID NO: 1 and / or SEQ ID NO: 2, and - 1-10 μM L-arginine and / or 1-10 μM L-lysine Including, Optionally, substantially free of neutralized deactivating agents and / or substantially free of neutralizing agents; Immunogenic compositions are also provided. As used herein, "1-10 μM" is specifically understood to be equivalent to "1 μM up to 10 μM." In particular, the immunogenic composition or mixture resulting from step (d) comprises 4-400 μg of recombinant PCV2 ORF2 per mL, preferably 8-200 μg of recombinant PCV2 ORF2 protein per mL.

[0043] "Substantially free of neutralizing agent" is understood to mean, among other things, that the immunogenic composition contains less than 150 nM of neutralizing agent. Thus, for example, an indication that "the immunogenic composition is substantially free of sodium thiosulfate" is understood to be equivalent to, among other things, the specification that "the immunogenic composition contains less than 150 nM of sodium thiosulfate." According to a further aspect, the present invention therefore relates to an immunogenic composition obtainable by the methods described and claimed herein, wherein the immunogenic composition or mixture obtained from step (d) is substantially free of sodium thiosulfate or contains less than 150 nM sodium thiosulfate. Furthermore, an immunogenic composition, in particular an immunogenic composition obtainable by the methods and claimed methods described herein, preferably by any such method including step (d), comprising: A recombinant PCV2 ORF2 protein, preferably comprising or consisting of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.5% or 100% sequence identity with the sequences of SEQ ID NO:1 and / or SEQ ID NO:2, and 0.1–1 μM L-tryptophan; and / or 0.1–3 μM L-glutamine; and / or 0.2-4 μM L-methionine; and / or 1-10 μM L-arginine; and / or 1-10 μM L-threonine; and / or 1-15 μM L-lysine An immunogenic composition comprising:

[0044] Preferably, the immunogenic compositions described and claimed herein comprise: 0.1-0.7 μM L-tryptophan; and / or 0.7–2.4 μM L-glutamine; and / or 0.5 to 3.2 µM L-methionine; and / or 1.4 to 4.7 μM L-arginine; and / or 1.6 to 8.0 µM L-threonine; and / or 4.0-12.2 μM L-lysine Includes.

[0045] More preferably, the immunogenic compositions described and claimed herein comprise: 0.2-0.6 μM L-tryptophan; and / or 0.8–2.2 μM L-glutamine; and / or 0.8 to 2.2 µM L-methionine; and / or 1.6 to 4.3 μM L-arginine; and / or 2.4 to 6.3 µM L-threonine; and / or 4.4 to 11.2 μM L-lysine Includes. In a particularly preferred embodiment, the immunogenic compositions described herein and claimed herein contain 0.7-2.4 μM L-glutamine and 4.0-12.2 μM L-lysine, or preferably 0.8-2.2 μM L-glutamine and 4.4-11.2 μM L-lysine.

[0046] Preferably, the immunogenic compositions described and claimed herein are substantially free of sodium thiosulfate. According to a further preferred embodiment, there is provided an immunogenic composition, in particular an immunogenic composition obtainable by the methods and claimed methods described herein, preferably obtained by any such method comprising step (d), which, when subjected to two-dimensional (2D) ultra-performance liquid chromatography (UPLC) at room temperature, exhibits a ratio of the area of ​​peak A to the area of ​​peak B of 0.2 to 0.4 and / or a ratio of the area of ​​peak C to the area of ​​peak B of 0.5 to 0.9, wherein the 2D UPLC A first dimension chromatography having a system volume of 400 μL, - a sample containing an immunogenic composition is injected into an anion exchange column having a column volume of 0.1 mL, the column being packed with a quaternary amine-functionalized material suitable for binding virus-like particles; and - a multi-step gradient method starting with 100% 50mM Tris, pH 8 at 0 min, 97.5% 50mM Tris, pH 8 and 2.5% 50mM Tris, 2M NaCl, pH 8 at 1 min, and finally 10% 50mM Tris, pH 8 and 90% 50mM Tris, 2M NaCl, pH 8 at 2 min, through an anion exchange column at a flow rate of 0.6mL / min; A first dimension of chromatography is included, At a retention time of 2.67 minutes, a switch was made from the first dimension chromatography to the second dimension chromatography, and 50 μL of the eluate of the first dimension chromatography was passed through a size exclusion column having a column volume of 1.75 mL with a pore size of 450 Å at a flow rate of 0.3 mL / min; Chromatograms were recorded by monitoring the fluorescence emission at a wavelength of 330 nm and excitation at a wavelength of 280 nm; The area of ​​peak A is the peak area of ​​the highest peak in the chromatogram with a retention time of 0 to 1 minute, The area of ​​peak B is the peak area of ​​the highest peak at a retention time of 7 to 8 minutes in the chromatogram, The area of ​​peak C is the peak area of ​​the highest peak with a retention time of 8.5 to 9.5 minutes in the chromatogram. Immunogenic compositions are provided.

[0047] According to one preferred embodiment, the sample comprises the immunogenic composition diluted in 50 mM Tris, pH 8. For example, in the case where the sample comprises the immunogenic composition remaining after step (c), then the sample preferably comprises or consists of the immunogenic composition diluted 1:10 in 50 mM Tris, pH 8. Quaternary amine-functionalized materials suitable for binding virus-like particles are preferably macroporous and / or non-porous materials. Example 5 describes such a 2D UPLC method that can be used by those skilled in the art.In case of debatable results and in case of any doubt, the peak area ratios referred to herein refer to the ratios that are estimated / can be estimated by the method described in Example 5.

[0048] The term "immunogenic composition" refers to a composition of matter, including but not limited to at least one antigen, that induces a cellular and / or antibody-mediated immune response in a host against the antigen of interest. Typically, an "immune response" includes but is not limited to one or more of the following effects: production or activation of antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells and / or gamma-delta T cells, specifically against the antigen or antigens contained in the composition or vaccine of interest. Preferably, the host presents either a therapeutic or protective immune response, such that resistance to new infections is enhanced and / or the clinical severity of disease is reduced. In such cases, the immunogenic composition is a "vaccine". Such protection is demonstrated by either a reduction or absence of symptoms normally exhibited by an infected host, a more rapid recovery time and / or a reduction in viral titers in the infected host. The host described herein is, in particular, a mammal, preferably a pig.

[0049] In a further embodiment, the virucidal activity of the immunogenic composition produced by the methods and claimed herein is reduced by at least 10%, preferably at least 20%, compared to the corresponding immunogenic composition that has not been subjected to the concentration of step (b) and the continuous diafiltration of step (c) of the method. More preferably, the virucidal activity of the immunogenic composition is reduced by at least 50% compared to the corresponding immunogenic composition that has not been subjected to the concentration of step (b) and the continuous diafiltration of step (c). Even more preferably, the virucidal activity of the immunogenic composition is reduced by at least 70% compared to the corresponding immunogenic composition that has not been subjected to the concentration of step (b) and the continuous diafiltration of step (c). Even more preferably, the virucidal activity of the immunogenic composition is reduced by at least 90% compared to the corresponding immunogenic composition that has not been subjected to the concentration of step (b) and the continuous diafiltration of step (c).

[0050] For purposes of the present invention, the term "virucidal activity" means that a liquid, fluid, solution, composition, etc., inactivates or kills to some extent a live virus or live bacteria when said liquid, fluid, solution, composition, etc. is mixed with such live virus or live bacteria. Thus, a reduction of at least 10% in the virucidal activity of a liquid, fluid, solution, composition, etc. means that the viability of live virus or live bacteria is 90% higher in a liquid, fluid, solution, composition, etc., that has been subjected to any of the manufacturing methods described herein, compared to a liquid, fluid, solution, composition, etc., that has not been subjected to any such manufacturing method. In this context, "viability" particularly relates to the percentage of viruses or bacteria that maintain the ability to replicate in a host.

[0051] In certain preferred embodiments, the immunogenic compositions produced by the methods and claimed methods described herein have an immunogenic effect of at least 1 log TCID of live virus when the live virus or live bacteria is mixed with the immunogenic composition and incubated for at least 4 hours. 50More preferably, the immunogenic compositions produced by the methods described and claimed herein cause a loss of less than 0.9 log TCID 25 per mL of live virus when the live virus or live bacteria is mixed and incubated with the immunogenic composition for at least 4 hours. 50 Even more preferably, the immunogenic compositions produced by the methods described and claimed herein cause a loss of less than 0.7 log TCID 25 per mL of live virus when the live virus or live bacteria is mixed and incubated with the immunogenic composition for at least 4 hours. 50 More preferably, the immunogenic compositions produced by the methods described and claimed herein cause a loss of less than 0.5 log TCID 25 per mL of live virus when the live virus or live bacteria is mixed and incubated with the immunogenic composition for at least 4 hours. 50 Even more preferably, the immunogenic compositions produced by the methods described and claimed herein cause a loss of less than 0.3 log TCID 25 per mL of live virus when the live virus or live bacteria is mixed and incubated with the immunogenic composition for at least 4 hours. 50 or causing a loss of less than 0.3 log CFU per mL of viable bacteria.

[0052] TCID per mL 50 can be estimated by standard in vitro titer assays that allow for estimation of the amount of live virus. CFU per mL can also be determined by standard in vitro titer assays that allow for estimation of the amount of live bacteria. The term "per mL" preferably refers to 1 mL of fluid. Furthermore, it has been found that the immunogenic compositions obtainable by the methods described and claimed herein are particularly stable. According to certain preferred embodiments, the immunogenic compositions produced by the methods described and claimed herein have a shelf life of at least 2 years, preferably at least 3 years, the term "shelf life" as used herein meaning the period during which the product can be stored without the quality dropping below some certain minimum acceptable level. In particular, the present invention relates to an immunogenic composition which, after administration of one dose of the immunogenic composition, preferably in a pig, induces a protective immune response against the pathogen from which the amino acid sequence of the recombinant protein is derived, wherein the pathogen is preferably PCV2, and wherein said administration is preferably the administration of one dose of the immunogenic composition to the pig. In another aspect, the invention relates to an immunogenic composition as described and claimed herein, further comprising a live attenuated virus, preferably an attenuated Porcine Reproductive and Respiratory Syndrome (PRRS) virus, or a live attenuated bacterium.

[0053] For the purposes of the present invention, "live" virus or "live" bacteria refers to a virus or bacteria that is capable of replicating in a host. The preferred live virus and preferred live bacteria of the present invention are PRRSV and Mycoplasma hyopneumoniae, respectively. However, the terms live virus and live bacteria are not limited to PRRSV and Mycoplasma hyopneumoniae, respectively. In another aspect, the invention relates to the immunogenic compositions described and claimed herein, wherein the immunogenic composition further comprises an attenuated porcine reproductive and respiratory syndrome (PRRS) virus. The term "attenuated" particularly relates to viruses or bacteria that are cultivated in a way to reduce their pathogenicity and make them suitable for use as a vaccine. Thus, more specifically, "attenuated" relates to viruses or bacteria that are alive but that are preferably no longer capable of causing disease due to the reduction of their pathogenicity or toxicity by means of such specific cultivation.

[0054] Preferably, the immunogenic composition induces a protective immune response against PRRS virus, particularly in pigs, following administration of one dose of the immunogenic composition, preferably administration of one dose of the immunogenic composition to the pig. More preferably, the immunogenic composition induces a protective immune response against PCV2 and PRRS virus, particularly in pigs, following administration of one dose of the immunogenic composition, preferably administration of one dose of the immunogenic composition to the pig. In another aspect, the present invention also provides a kit comprising a container containing the immunogenic compositions described herein and the claimed immunogenic compositions, and further comprising at least one additional container containing at least one additional antigen, preferably selected from the group consisting of a live attenuated virus, preferably an attenuated PRRS virus, and a live attenuated bacterium. According to a further aspect, there is provided a kit comprising a container containing the immunogenic compositions described herein and the claimed immunogenic compositions.

[0055] The immunogenic compositions and claimed immunogenic compositions described herein and / or the kits and claimed herein may further comprise at least one additional antigen, preferably a viral or bacterial antigen, even more preferably a viral or bacterial antigen from at least one other pathogen causing disease in pigs. The additional antigen may be any one of the antigens disclosed in International Patent Application WO 2007 / 094893, the contents and teachings of which are incorporated herein by reference. Briefly, the additional antigen may be an antigen of any other pathogen causing disease in pigs. Preferably, the "any other pathogen causing disease in pigs", or at least one other pathogen causing disease in pigs, is Actinobacillus pleuropneumoniae (1); adenovirus (2); alphavirus, such as Eastern equine encephalitis virus (3); Bordetella bronchiseptica (4); Brachyspira species (5), preferably B. hyodysentheriae (6); B. piosicoli (7); Brucella suis, preferably biovars 1, 2 and 3 (8); classical hog cholera virus (9); Clostridium species (10), preferably Cl. difficile (11), Cl. perfringens types A, B and C (12), Cl. novi (13), Cl. septicum (14), Cl. tetani (15); coronavirus (16), preferably For example, porcine respiratory coronavirus (17); Erysipelothrix rhusioensis suis (18); Erysipelothrix rhusiopathiae (19); Escherichia coli (20); Haemophilus parasuis, preferably subtypes 1, 7 and 14 (21); hemagglutinating encephalomyelitis virus (22); Japanese encephalitis virus (23); Lawsonia intracellularis (24); Leptospira species (25), preferably Leptospira australis (26); Leptospira canicola (27); Leptospira grippotyphosa (28); Leptospira icterohaemolagicae (29); and Leptospira interrogans (30); Leptospira pomona (31); Leptospira tharassovi (32); Mycobacterium species (33), preferably Mycobacterium avium (34), Mycobacterium intracellulare (35) and Mycobacterium spp.bovis (36); Mycoplasma hyopneumoniae (37); Pasteurella multocida (38); porcine cytomegalovirus (39); porcine parvovirus (40); porcine reproductive and respiratory syndrome (PRRS) virus (41); pseudorabies virus (42); rotavirus (43); Salmonella spp. (44), preferably S. typhimurium (45) and S. cholerae suis (46); Staphylococcus hyicaus (47); Staphylococcus spp. (48 ), preferably selected from the group consisting of Streptococcus species (49), preferably Streptococcus suis (50); swine herpes virus (51); swine influenza virus (52); swinepox virus (53); swinepox virus (54); vesicular stomatitis virus (55); vesicular ecchymoses of pigs virus (56); Leptospira hardjo (57); Mycoplasma hyoscynoviae (58), and / or combinations thereof.

[0056] Most preferably, the immunogenic compositions and claimed immunogenic compositions described herein, and / or the kits and claimed herein, further comprise one or more attenuated or inactivated non-PRRSV pathogens, or antigenic materials thereof. In another aspect, the invention relates to the immunogenic compositions as described and claimed herein, and / or the kits as described and claimed herein, for use as a medicament, preferably as a vaccine.

[0057] In a further aspect, the present invention relates to a method for the production of a porcine ovarian cancer cell line comprising the steps of: - induce a protective immune response against at least one pathogen, and / or - reducing one or more clinical signs of infection with at least one pathogen The present invention relates to the immunogenic compositions and claimed immunogenic compositions described and claimed herein, and / or the kits and kits described and claimed herein, for use in the methods. The present invention relates to a method for the treatment of an animal comprising administering to said animal - induce a protective immune response against at least one pathogen, and / or - reducing one or more clinical signs of infection with at least one pathogen Further provided is a method, wherein the immunogenic compositions described and claimed herein are administered to an animal, in particular a pig.

[0058] According to a still further aspect, the present invention relates to a method for producing a porcine ovarian cancer cell line comprising the steps of: - induce a protective immune response against at least one pathogen, and / or - reducing one or more clinical signs of infection with at least one pathogen The present invention relates to the use of the immunogenic compositions described and claimed herein for the manufacture of a medicament for the treatment of a cancer. Preferably, the at least one pathogen is selected from the group consisting of PCV2, PRRSV, a non-PRRSV pathogen, and combinations thereof. As used herein, the term "non-PRRSV pathogen" particularly relates to a pathogen selected from Mycoplasma hyopneumoniae, pseudorabies virus, swine influenza virus, porcine parvovirus, transmissible gastroenteritis virus, Escherichia coli, Erysipelothrix rhusiopathiae, Bordetella bronchiseptica, Salmonella cholerae suis, Haemophilus parasuis, Pasteurella multocida, Streptococcus suis and Actinobacillus pleuropneumoniae.

[0059] As used herein, the term "reducing one or more clinical signs" is intended to mean that any such signs are reduced in incidence or severity in animals receiving the immunogenic composition compared to animals in a "control group," particularly when both are infected or challenged with a pathogen from which the immunogenic active ingredient in the immunogenic composition is derived, and the control group does not receive the immunogenic composition. In this context, the term "reduced" means a reduction of at least 10%, preferably 25%, even more preferably 50%, and most preferably 100% in the group receiving the immunogenic composition compared to the control group. As used herein, "clinical signs" refers to symptoms of infection resulting from a pathogen that are directly observable in a live animal, such as symptoms. Representative examples will depend on the pathogen selected, but can include such things as runny nose, lethargy, coughing, elevated fever, reduced weight gain or weight loss, dehydration, diarrhea, swelling, lameness, and the like. As used herein, a "protective immune response" refers to a reduction in the incidence or severity of clinical, pathological or histopathological signs or symptoms consistent with infection from a pathogen of interest, including complete prevention of such signs or symptoms. EXAMPLES

[0060] Example 1 PCV2 ORF2 Protein Production – Upstream Processing PCV2 ORF2 protein is produced in baculovirus-infected SF+ cells in a process corresponding to that described in Examples 1 to 3 of WO 2006 / 072065. This process is summarized below. PCV2 ORF2 baculovirus vector is produced in a bioreactor. Pre-sterilized or sterile filtered media is added to the bioreactor. Media with SF+ cells from which the expression culture originates is added. Cells are inoculated at the same time as inoculating with the PCV2 ORF2 seed (co-infection). Temperature is maintained at 27 ± 2°C and pH is monitored while the virus is growing. Dissolved oxygen (DO) is controlled by sparging with purified compressed air and oxygen (O2). The harvest window occurs 6-8 days after virus infection, when a harvest criterion of ≤20% cell viability is reached. At the time of harvest, the PCV2 ORF2 antigen fluid is clarified using two sets of filters: a pre-filter with a 2.0-15.0 μm pore size and a final filter with a 0.8-1.0 μm pore size. The filtered harvest fluid is collected in a tank. For baculovirus inactivation, 5 mM BEI is added up to 96 hours before neutralization. To neutralize the remaining BEI, 5 mM sodium thiosulfate is added to the neutralized BEI. The addition of sodium thiosulfate occurs 72-96 hours after inactivation.

[0061] PCV2 ORF2 Protein Production – Downstream Processing Laboratory Scale A 3 L sample obtained from the upstream process described above was taken in the laboratory and concentrated to 300 mL based on volume to reach a 10-fold concentration. After concentration, the antigen was then diafiltered using 3 L of P-saline, a buffer consisting of 0.85% NaCl dissolved in water and adjusted to a pH of 6.8-7.0. Laboratory-scale concentration and continuous diafiltration were performed in a biosafety cabinet or BSC using the following: one Sartoslice Ultrafilter (UF) cassette filter (PES, 200 cm 2 The procedure was performed at room temperature (27±2°C) using a tangential flow filter (TFF) system consisting of a TFF System Sartoflow Slice 200 benchtop system constructed with a filtration area of ​​1000 nm and a cut-off of 300 kD.

[0062] Prior to UF / DF, the TFF system was rinsed with 1M NaOH (at least 1 hour) and the cassette was chemically sterilized with 0.1M NaOH overnight. The sterilized system and filter were then placed in the BSC for aseptic assembly and the inlet, outlet and permeate lines were connected. The inlet line was connected to the concentrated inactivated antigen while the permeate line was connected to the waste bottle. The outlet line originated from the filtration unit and returned to the holding bottle. The filters were equilibrated using sterile PBS (10L per filter). The product was concentrated by ultrafiltration to 10x the original antigen volume on a volumetric basis. The flow rate through the filter was varied as the antigen was processed by concentration. At the small scale level, the flow rate ranged and was maintained by applying back pressure to the system. Diafiltration was performed at 10x the ultrafiltration volume against P-saline, and P-saline was added at a rate equal to the permeate flow. The final concentrated and diafiltered antigen was stored at 2-8°C.

[0063] Utilizing HPLC quantification methods, it was found that this process removed ≧99% of any residual insect cell medium (ExCell 420 medium) and reduced neutralized BEI and residual sodium thiosulfate to undetectable levels. Initial experiments also show that in the context of the manufacture of the immunogenic compositions described herein, the use of flat sheet filters for TFF systems has the advantageous effect of significantly reducing storage costs, as smaller storage bags / barrels (200 L) are required to be utilized, as compared to hollow fiber filters. In addition, it has been found that the use of flat sheet filters significantly reduces production time as well as labor for collection.

[0064] Operation Scale In an operational or large scale process (see Figure 1), approximately 4000 L is used, obtained from the upstream process described above. The system is designed so that the antigen-containing solution in the inactivation tank can be sent directly to the downstream processing (DSP) tank at a continuous feed rate until the inactivation tank is empty. The DSP tank is the storage location for the antigen in the process and for the final antigen preparation. The programmed mass of this tank controls the entire system in that once a certain programmed mass is reached, the process moves forward. Additionally, this tank is where the final antigen product is controlled and collected in a 200L barrel for storage. The DSP tank is connected to a microfiltration (MF) unit. After sending the antigen-containing solution from the DSP tank to the MF unit, the antigen is concentrated and the retentate is sent back to the DSP tank, while the permeate is sent to waste.

[0065] The process starts by taking the mass of the inactivation tank containing the (intermediate) product obtained from the upstream processing. A 10-fold concentration is calculated based on the initial mass of the tank and programmed into the system's operating unit for the MF unit. Once the concentration starts, the system starts the transfer line between the inactivation tank and the DSP tank, which opens or closes based on the tank mass. When the inactivation tank is empty, the system starts the tank to close the transfer line to the DSP tank. After the inactivation tank is empty, another transfer line is activated for saline to add to the just emptied inactivation tank. The saline is also monitored by mass, since the desired amount of saline to be transferred to the DSP tank is the original mass taken and is used for the calculation of concentration. To further concentrate the retentate, it is fed back to the DSP tank and passed further through the MF unit again until the mass of the retentate is reduced to a programmed value indicating the desired concentration.

[0066] The MF unit is unique in that it can support both microfilter and ultrafiltration cassettes. A total of six ultrafiltration sartocubes (21 m per cube) were used for this procedure. 2 ) is accommodated in the MF unit. This surface area was selected after an expansion evaluation of the system. There is a feed line from the DSP tank to the MF unit, a retentate line feeding back to the DSP tank, and a permeate-waste transfer line. The unit is programmed to run at 0 psi permeate pressure while maintaining a recirculation of 20-35 psi with a target transmembrane pressure (TMP) of 29 psi. The recirculation pump runs at 100% capacity until the hold mode is activated. These parameters are the targets during the concentration and diafiltration process. The MF unit activates when the DSP tank is at the set mass to start the concentration step and keeps the antigen in circulation until the set 10x concentration is achieved. Once the 10x concentration is finished, the MF unit switches to hold mode to circulate the antigen at a reduced pressure and speed while the inactivation tank is filled with P-saline. After the P-saline reaches the desired volume in the inactivation tank, which is the original mass at the start, the system starts the diafiltration process by starting the flow of P-saline into the DSP tank and the MF unit increases back up to the target range.

[0067] Once the inactivation tank is empty, the valve between the inactivation tank and the DSP tank is closed and the antigen is further processed through the MF unit until 10x diafiltration is achieved. The concentrated and diafiltered antigen is in the DSP tank and is pumped into the antigen collection barrels for final storage. These barrels are stored at 4±2°C until vaccine formulation. The products obtained from the above upstream and downstream processing were then finally mixed with carbomer solution (adjuvant) and saline (P-saline). This research animal product is also referred to hereinafter as Research Animal Product (IVP) No. 1 or "IVP1", respectively. In conclusion, with regard to the new process described herein, the particular advantages observed were time savings, reduced handling, and cooler storage / space savings.

[0068] Example 2 Stability studies in use Abstract Utilizing an optimized protocol, a study was designed to investigate the in-use stability of a research animal product (lyophilisate) containing a modified live PRRS virus after reconstitution with Research Animal Product (IVP) No. 1 (IVP1) of Example 1 (said research vaccine product is also referred to hereinafter as "IVP2"). An overview of the investigational veterinary products (IVPs) used is presented in Table 1. The evaluation included two batches of IVP1, one manufactured at high potency and one manufactured at normal / standard release potency, as well as two batches of IVP2. All tested investigational veterinary products (IVPs) (see Table 1) were stored at 5°C ± 3°C until use for the study. In this study, each batch of IVP2 vaccine was reconstituted with IVP1 and PRRS virus titers were tested 0, 2 and 4 hours after reconstitution. For each IVP2 batch, the PRRS virus titer after reconstitution at time 0 was similar regardless of whether the lyophilisate was reconstituted with WPBS (washed phosphate-buffered saline (=phosphate-buffered saline wash solution)) or with IVP1 and regardless of scientist. In addition, the test results showed a non-negligible change in virus titer after 2 and 4 hours of incubation at room temperature.

[0069] The results of the study support the linked use of IVP1 and IVP2. 1. Introduction A study was designed to demonstrate the in-use stability of IVP2 after reconstitution with IVP1, a lyophilized modified live vaccine (MLV), and IVP1, produced as described above. The results of the stability evaluation during use of IVP2 after reconstitution with IVP1 are shown below. 2. Research goals / objectives The goal of this in-use stability study was to investigate the stability of the IVP2 vaccine after reconstitution with IVP1.

[0070] 3.Material Two batches of each of the two vaccines being evaluated, and one batch of the diluent control used in the study, were used and the composition of the test materials are listed in Table 1. Table 1. Test material composition [Table 1]

[0071] 4. Study Design Each batch of the IVP2 vaccine was tested in all possible combinations with each batch of the subunit vaccine (IVP1). Table 2.1 lists all eight vaccine combinations evaluated in this study. Table 2.1 List of evaluated IVP combinations [Table 2]

[0072] Samples were tested for PRRS virus titer immediately after complete reconstitution and pooling at time zero (T0). Pooled samples were stored in the dark at room temperature and tested again at 2 hours (T2) and 4 hours (T4). Samples were prepared on the same day as testing. Each sample was tested in two different sessions, which were performed on different days. Testing was performed by two scientists. Scientist 1 tested sample numbers 1-4 using IVP2 batch No. 1, and Scientist 2 tested sample numbers 5-8 using IVP2 batch No. 2. Each scientist tested two samples and an in-use stability control per session. Controls consisted of IVP2 reconstituted in sterile washed phosphate buffered saline (WPBS) using the same volumes and time intervals as indicated for the corresponding test samples.

[0073] 5. Method 5.1 Recovery method For each sample (as described in Table 2.1), each of the three vials of IVP2 was reconstituted with 10 mL of IVP1. For each control, each of the three vials of IVP2 was reconstituted with 10 mL of WPBS. After reconstitution was completed, the contents of each of the three vials described above were pooled before starting the test.

[0074] 5.2 Test Method Day 3 A cell culture suspension of AK-MA104 cells was prepared in MEM supplemented with 10% FBS and gentamicin sulfate. 100 μL of cell culture was seeded into each well of a 96-well microtiter plate. The plate was closed with a lid and kept in an incubator for 3 days. Day 0 Sample preparation Samples of IVP1 and the positive control were used to resuspend a lyophilized sample of IVP2. The three vials were pooled and the pool was titrated in triplicate. titration As a dilution medium, MEM supplemented with 2% FBS and gentamicin sulfate was used to dilute the samples.

[0075] inoculation Sample wells were inoculated with 100 μL of diluted sample and negative control wells were inoculated with 100 μL of medium. incubation This was incubated in an incubator for 8 days. +8th day Reading and Calculation Final cytopathic effect (CPE) readings of the plates were taken under an inverted microscope and the raw data were converted to titers per mL dose by adding 1.0 log.

[0076] Titers were calculated according to the Reed and Muench method (Reed, LJ; H. Muench, (1938). "A simple method of estimating fifty per cent endpoints." Am. J. Hygiene, 27: 493-497) and expressed as log10 TCID50 per mL dose. The titer of each sample at each test interval was reported as the arithmetic mean titer per dose of independent titrations of two consecutive valid test sessions with three independent titrations per session. 6. Acceptance Criteria Viral titers of IVP2 obtained after 0 h (T0), 2 h (T2) and 4 h (T4) of reconstitution with WPBS diluted control are higher than or equivalent to the given / previously determined minimum immunizing dose (MID).

[0077] 7.Results A summary of the mean titers obtained for the test samples and each control at each incubation time (T0, T2 and T4) and the corresponding titer differences relative to time 0 (T0-T0, T2-T0, T4-T0) are shown in Tables 3 and 4, respectively. Table 3. Mean viral titers [Table 3]

[0078] Table 4. Difference in mean viral titer from T0 [Table 4]

[0079] 8. Discussion Data obtained in this study demonstrated that viral titers of IVP2 rehydrated in standard diluent (WPBS) were all above the MID and remained stable during 4 h of incubation at room temperature. These results are consistent with known and previously determined product properties of IVP2 and confirm the validity of this in-use stability evaluation of IVP2 of the modified live PRRS virus vaccine in conjunction with IVP1.

[0080] The results obtained for both batches of IVP2 tested with either of the two batches of IVP1 at T0 show comparable viral titers to those obtained with the respective controls. The variability observed between the control and test samples is within the assay variability seen in the respective assay validation studies. After reconstitution with either of the two batches of IVP1 at T2 and T4, the results obtained for both batches of IVP2 showed stable virus titers with no minimal (≦0.12 log10 TCID50) decrease in titer. 9. Conclusion The results of this study support the associated use of IVP1 and IVP2, and the 4 hour in-use stability of IVP2 when mixed with IVP1.

[0081] Example 3 A) Development of immunity to PRRSV challenge The goal of this study was to investigate whether the development of immunity to IVP2 is affected 3 weeks after vaccination when used in association with IVP1. For this purpose, the IVP2 vaccine was reconstituted to the minimum immunizing dose (MID) with IVP1 produced in high potency, and this combination of IVP1 and IVP2 is hereafter also referred to as "IVP1 / IVP2". The study included 52 3-week-old piglets from a production farm in Germany. The primary parameter of the study was lung lesions at necropsy 10-12 days after challenge. None of the animals contained positive antibodies or antigens for PRRSV. Up until challenge with PRRSV (21 days after vaccination), no animals in the control group seroconverted or tested positive for PRRSV antigens. IVP1 / IVP2 vaccinated animals had a 52.1% reduction in median embryo lesion scores compared to control animals. Furthermore, the ADWG results support the efficacy of the combination of IVP1 and IVP2, as IVP1 / IVP2 vaccinated animals had a statistically significant higher ADWG of 164 g per day after challenge compared to controls.

[0082] In animals vaccinated with IVP1 / IVP2, the first PRRSV antibodies were detected 14 days after vaccination (D14). The number of PRRSV antibody-positive animals was not statistically significantly different between the two groups 1 week after challenge, but the levels of antibodies were significantly higher in the vaccinated group compared to the control group. 10-12 days after challenge, all animals were positive for PRRSV antibodies, and there was no statistically significant difference in the levels of antibodies between the groups. In conclusion, we demonstrated that the development of immunity to IVP2 3 weeks after vaccination was not negatively affected when used in conjunction with IVP1. B) Development of immunity to PCV2 challenge The goal of this study was to evaluate the development of immunity against IVP1 when used in conjunction with IVP2. To this end, the IVP2 vaccine was restored to target a maximum immunizing dose by adding IVP1 formulated to target a minimum potency; this mixture of IVP1 and IVP2 is also referred to hereinafter as "IVP2 / IVP1".

[0083] The study included 60 Caesarean-derived colostrum-deficient (CDCD) pigs that were seronegative for PCV2, 30 of which were vaccinated with IVP2 / IVP1 at 3 weeks of age (i.e., study day 1 (D0)) and 30 (control group) received sterile diluent (water for injections) followed by a virulent PCV2 challenge on D15. Vaccination with IVP2 / IVP1 resulted in a significant increase in pigs positive for PCV2 serology. Within one week of challenge, 28 pigs (93%) from the IVP2 / IVP1 group seroconverted to positive levels, whereas 0 / 30 (0%) pigs from the control group were positive. Animals from the control group started seroconverting to positive levels 13 days after challenge. At D21, D28 and D35, the IVP2 / IVP1 group had a significantly higher percentage of pigs with positive PCV2 titers compared to the control group. By D42, the IVP2 / IVP1 group was 100% serologically positive for PCV2, whereas the control group was 92% positive. In assessing the primary outcome parameters, vaccination with IVP2 / IVP1 significantly reduced lymphatic depletion, lymphatic inflammation and PCV2 lymphatic colonization compared to the control group. Furthermore, the overall level of histological lesions was more severe in the control group with 23 / 30 pigs (76.7%) having a moderate to severe score in at least one category of lymphatic lesion assessment, whereas only 2 / 30 pigs (6.7%) of vaccinated pigs had a moderate lymphatic lesion score and none were severe.

[0084] In conclusion, IVP1, formulated with minimal potency and used in association with IVP2, provided effective active immunity of 3-week-old CDCD pigs 15 days after vaccination when challenged with virulent PCV2. In addition, in a further vaccination-PCV2 challenge study, monovalent IVP1 was compared to the respective product not subjected to downstream processing according to Example 1 (herein referred to as "IVP0") and it was found that IVP1 was at least as effective as the established IVP0. C) Duration of immunity to PCV2 challenge The goal of this study was to evaluate the duration of immunity of IVP1 when used in conjunction with IVP2. To this end, the IVP2 vaccine was restored to target a maximum immunizing dose by adding IVP1 formulated to target a minimum potency. As mentioned above (section B), this mixture of IVP1 and IVP2 is also referred to herein as "IVP2 / IVP1".

[0085] The study included 60 Caesarean-derived colostrum-deprived (CDCD) pigs that were seronegative for PCV2, at 3 weeks of age (i.e., study day 1 (D0)), of which 30 were vaccinated with IVP2 / IVP1 and 30 (control group) received sterile diluent (water for injections) followed by a virulent PCV2 challenge on D119 (duration of immunization of 17 weeks). As a result, animals vaccinated with IVP2 / IVP1 had significantly reduced frequencies of lymphodepletion, lymphatic colonization, and viremia compared to control animals following PCV2 challenge. In addition, animals vaccinated with IVP2 / IVP1 had significantly reduced PCV2 viral loads at all sampling dates, and although virus levels were not considered to be disease-causing viremia at any sampling point ((rt-PCR) test ≥ 1.0 × 10 4 PCV2 genome equivalents), control animals were viremic at a median of 28 days after challenge with PCV2. Vaccination with IVP2 / IVP1 resulted in a significant increase in positivity for PCV2 serology from vaccination (D0) to D133 (2 weeks after challenge with PCV), historically a strong indicator of efficacy, in pigs. The majority of the control group (81%) did not test positive for PCV2 serology until D140 (3 weeks after challenge with PCV2). These results indicate that the duration of immunity of IVP1 over the 17 weeks following vaccination is not negatively affected when used in conjunction with IVP2.

[0086] D) Duration of immunity to PRRSV challenge The goal of this study is to investigate whether the duration of immunity of IVP2 is affected 6 months after vaccination when used in conjunction with IVP1. For this purpose, the IVP2 vaccine was reconstituted to the minimum immunizing dose (MID) with IVP1 produced in high potency. As mentioned above (section A), this mixture of IVP1 and IVP2 is also called "IVP2 / IVP1". The study included 70 3-week-old piglets from a production farm in Germany, with no animals containing positive antibodies or antigens for PRRSV. The challenge was performed 182 days after vaccination (duration of immunity of 6 months). The main parameter of the study was the lung lesions at necropsy 10-12 days after the challenge. As a result, animals vaccinated with IVP2 / IVP1 had reduced embryonic pathology compared to control animals following PRRSV2 challenge, indicating that the duration of immunity of IVP2 6 months post-vaccination is not negatively affected when used in conjunction with IVP1.

[0087] Example 4 Thiosulfate concentration analysis Abstract Binary ethyleneimine was used as an inactivating agent in the production of baculovirus recombinant vaccines as described in Example 1. After inactivation with BEI, sodium thiosulfate is added to neutralize residual BEI. After inactivation of PCV2 antigen fluid with 5 mM BEI, low concentrations of BEI (typically on the order of 0.3 mM) remain detectable. The EU Pharmacopoeia states that BEI must not remain in the antigen after inactivation. The EU Pharmacopoeia considers detection of residual sodium thiosulfate after inactivation to be acceptable to ensure that no toxic levels of BEI remain in the antigen. Therefore, since 5 mM BEI is initially added at the beginning of the inactivation step, 5 mM thiosulfate was utilized to neutralize the BEI in the activation step. Because a significant amount of BEI is consumed in reaction with nucleic acids during inactivation, and because thiosulfate and BEI react with a 1:1 stoichiometry, excess residual thiosulfate remains after neutralization.

[0088] Thiosulfate testing was carried out on antigen fluids prepared according to Example 1 as described below. The data from this experiment show that the method described in Example 1 unexpectedly reduces thiosulfate levels already below the detection limit of the method in 10-fold concentrated diafiltered antigen fluids. material and method Quantitative determination of sodium thiosulfate in antigenic fluids by high performance liquid chromatography(HPLC). A. Reagents and Materials 1. Sodium Thiosulfate Pentahydrate, Reagent Grade of Known Purity 2. Purified water - resistivity of 18MΩ or higher is used 3. Acetonitrile (ACN), HPLC grade 4. Glacial acetic acid, HPLC grade 5. Tetrabutylammonium hydroxide (TBA), reagent grade 6. HPLC Column / Guard - Thermo Scientific, ODS-2 Hypersil, 150 x 4.6 mm, 5 μm with suitable guard, ODS-2 Hypersil-2, 10 x 4.6 mm, 5 μm, or equivalent

[0089] B. Solution 1.20% acetic acid solution Combine 10 mL of glacial acetic acid and 40 mL of purified water and mix thoroughly. 2. Eluate Combine 750 mL of purified water and 20 mL of TBA and mix thoroughly. Adjust the pH of this solution to 4.0-5.0 by adding 20 mL of 20% acetic acid solution, then combine with 250 mL of acetonitrile. Mix thoroughly and filter through a 0.2 μm nylon filter before use. 3. Diluent Combine 800 mL of purified water and 200 mL of acetonitrile, mix thoroughly and store at ambient conditions. C. Thiosulfate Reference Standard Solution 1. Sodium thiosulfate stock solution (5 mM) Weigh out 1.2 g of sodium thiosulfate pentahydrate reference material and transfer to a 1000 mL Class A volumetric flask containing purified water and mix thoroughly by inversion. 2. 100% Working Standard Solution (100%WA) Transfer 2 mL of sodium thiosulfate stock solution to a 50 mL Class A volumetric flask. Dilute to volume with diluent and mix thoroughly by inversion. 3. 50% Linearity Standard Solution (50% LS) Transfer 1 mL of sodium thiosulfate stock solution to a 50 mL Class A volumetric flask. Dilute to volume with diluent and mix thoroughly by inversion. 4. 150% Linearity Standard Solution (150% LS) Transfer 3 mL of sodium thiosulfate stock solution to a 50 mL Class A volumetric flask. Dilute to volume with diluent and mix thoroughly by inversion. 5. Limit of Quantitation (LOQ) Transfer 1 mL of 50% LS to a 20 mL Class A volumetric flask. Dilute to volume with diluent and mix thoroughly by inversion.

[0090] D. Test solution of PCV2 inactivated antigen fluid Transfer 500 μL of inactivated antigen fluid into a 10 mL class A volumetric flask. Dilute to volume with diluent and mix thoroughly by inversion. Filter the sample through a 0.2 μm nylon syringe filter into an HPLC vial and discard the first 2-3 mL. This is a 20x dilution of antigen fluid. E. HPLC Chromatography Equipment and Conditions 1.Flow rate: 1.2mL / min 2. Injection volume: 10 μL 3. Columns: Thermo Scientific, ODS-2 Hypersil, 150×4.6 mm, 5 μm with suitable guard, ODS-2 Hypersil-2, 10×4.6 mm, 5 μm 4.Temperature: Sample tray temperature: 20±2℃ Column oven temperature: 25±2℃ 5. Detection: 230nm 6. Typical Analysis Sequence - Waste Dilution Fluid - Diluent; 1 injection - Blank: 1 injection (baseline monitoring, no injection) - Calibration standard; 6 injections of 100% WS - Linearity standards; 2 injections each of 50, 100 and 150% solutions - LOQ standard; 2 injections - Test solutions and check standards; 2 injections per test solution and 2 injections of 100% WS as check standard during the injection and end of analysis for a maximum of 16 samples. 7. Thiosulfate retention time: approx. 7 minutes

[0091] F. Calculate Sodium Thiosulfate Content – ​​Calculate the thiosulfate content as mM using the following formula: Thiosulfate (mM) = TS / WS x C x 20 During the ceremony, TS - the average of the thiosulfate peak areas of two injections of the test solution preparation; WS - average thiosulfate area response of six 100% WS injections; C - concentration of the working standard solution, mM (0.2 mM); 20 - Dilution factor of the test solution.

[0092] result Several antigen fluids prepared as described in Example 1 were analyzed, and paired samples were (i) after neutralization of BEI with sodium thiosulfate but before downstream processing; and (ii) after downstream processing (i.e., 10-fold concentration and diafiltration) but before mixing with the adjuvant and P-saline; and the sodium thiosulfate concentration was determined. We unexpectedly found that while sample (i) taken after BEI neutralization but before downstream processing still had sodium thiosulfate concentrations in the millimolar range, in sample (ii) taken after downstream processing but before mixing with adjuvant and P-saline, sodium thiosulfate was not detectable at or above the detection limit (3 μM).

[0093] Example 5 Analysis of antigenic fluids by two-dimensional UPLC A two-dimensional UPLC method based on anion exchange and size exclusion chromatographic separation was utilized for the analysis of virus-like particle (VLP)-containing antigenic fluids obtained from the process described in Example 1. The first phase chromatography used is anion exchange chromatography, where negatively charged species bind to the column (0-2.67 min in the chromatogram) and are then eluted by increasing concentrations of sodium chloride in a gradient. The second phase chromatography used is size exclusion (2.67-15 min in the chromatogram), where the valve is switched to allow the injection from the first dimension of anion exchange onto the size exclusion column, which elutes the mixture of anion exchange eluents.

[0094] material and method A. Reagents and Materials 1. 1M Tris in water, pH 7.4, molecular biology grade 2. 2.5 M NaCl in water, molecular biology grade 3. Purified water - resistivity of 18MΩ or higher is used 4. Anion exchange column - column volume 0.1 mL, with quaternary amine functionality suitable for binding of VLPs (macroporous or non-porous material) 5. Size Exclusion Column – Column volume 1.75 mL, pore size 450 Å 6.10N NaOH, reagent grade B. Solution 1. 50 mM Tris, pH 8: Combine 50 mL of 1 M Tris, pH 7.4 with 900 mL of water. Adjust to pH 8 with 10 N NaOH, dilute to 1000 mL, and filter through a 0.22 μm filter. 2. 50 mM Tris, 2 M NaCl, pH 8: Combine 50 mL of 1 M Tris, pH 7.4 and 400 mL of 5 M NaCl with 300 mL of water. Adjust to pH 8 with 10 N NaOH, dilute to 1000 mL and filter through a 0.22 μm filter.

[0095] C. PCV2 in process reference solution 1. Filter the resulting antigen fluid prior to inactivation through a 0.22 μm PES filter. D. Test solution of PCV2 pre-inactivated or inactivated antigen fluid 1. Filter the antigen fluid through a 0.22 μm PES filter. E. 2D-UPLC Chromatography Equipment and Conditions 1. First Dimension Chromatography a.Flow rate: 0.6mL / min b. Injection volume: 20 μL c. System volume: 400 μL d. Column: 0.1 mL column volume, quaternary amine column e. Temperature: room temperature f. Detection: Fluorescence emission (excitation 280 nm and emission 330 nm) g. Gradient: First, a multiple step gradient method from 100% 50 mM Tris, pH 8 at 0 min, to 97.5% 50 mM Tris, pH 8 and 2.5% 50 mM Tris, 2 M NaCl, pH 8 at 1 min, and finally 10% 50 mM Tris, pH 8 and 90% 50 mM Tris, 2 M NaCl, pH 8 at 2 min is run through an anion exchange column at a flow rate of 0.6 mL / min.

[0096] 2. Second Dimension Chromatography a.Flow rate: 0.3mL / min b. Injection from the first dimension: 50 μL c. Column: 1.75 mL column volume, size exclusion column with 450 Å pore size d. Detection: Fluorescence emission (excitation 280 nm and emission 330 nm) e. Isocratic gradient: 95% 50 mM Tris, pH 8: 5% 50 mM Tris, pH 8, 2 M NaCl 3. Typical Analysis Sequence a. Five injections of two-fold serial dilutions of the PCV2 reference solution b. 1 injection - 15 samples c. Five injections of two-fold serial dilutions of the PCV2 reference solution 4. Switching time from first to second dimension: 2.67 min (this switching time depends on the volume of the system, in this case 400 μL) 5. The peak ratios listed below were calculated by integrating the peak areas and taking the ratio of said peak areas.

[0097] Using such two-dimensional UPLC to analyze the VLP(ORF2) product obtained from the baculovirus expression system, distinct prominent peaks were seen in the resulting chromatogram. the peak between 0 and 1 min in the chromatogram, representing constituents that do not bind to the anion exchange column at pH 8 (i.e. constituents that are neutral or cationic at pH 8, e.g. positively charged proteins and / or cell culture components), hereinafter also referred to as "Peak A". - the peak at 7-8 min in the chromatogram, representing the VLP(ORF2) antigen of interest, also referred to hereinafter as "peak B". - the peak between 8.5 and 9.5 min in the chromatogram, representing constituents ranging in size from IgG (150 kDa) to uracil (0.1 kDa), also referred to hereinafter as "peak C".

[0098] It was found that 10-fold dilutions of samples taken after downstream processing as described in Example 1 (i.e., 10-fold concentration and continuous diafiltration) showed, on average, a 99.2% reduction in peak A and a 42% reduction in peak C compared to the upstream process samples. Additionally, a reduction in peaks A and C was found, but an increase in peak B was observed. Surprisingly, the peak ratios (Peak A / Peak B and Peak C / Peak B) for samples taken after downstream processing as described in Example 1 were found to be only 0.3 for Peak A / Peak B (standard deviation of 0.1 for 3 replicates) and 0.7 for Peak C / Peak B (standard deviation of 0.2 for 3 replicates). It was further found that there was no aggregation or increased presence of larger sized species in solution in samples taken after downstream processing (i.e., 10-fold concentration and continuous diafiltration) as described in Example 1. This observation was surprising since one would have expected to see a significant increase in larger species in such concentrated samples in size exclusion chromatography (SEC) due to the dependence of aggregation on concentration.

[0099] Example 6 Free amino acid quantification Conventional methods for quantifying free amino acids in insect culture medium and post-consumer culture medium were applied to determine the concentration of amino acids in antigenic fluids produced as described in Example 1. Briefly, proteins in the samples are precipitated with trichloroacetic acid (TCA) and removed by centrifugation. The supernatant from the TCA precipitation is neutralized with sodium hydroxide and norvaline is added as an internal standard. The samples are then derivatized with o-phthaldialdehyde (OPA) and mercaptopropionic acid (MPA) in the injection loop of the UHPLC before being injected onto a reversed-phase column for quantification by UV absorption at 338 nm. Based on the chromatograms for the standard reference samples (external standards), the concentrations of the 20 common amino acids (i.e., the 20 genetically encoded amino acids) contained in the antigen fluid were determined.

[0100] Measuring the amino acid concentrations of several samples taken after downstream processing described in Example 1 (i.e. 10-fold concentration and continuous diafiltration) and taking into account the usual subsequent dilution of said samples by adding adjuvant and P-saline, it was found that the characteristic concentrations of certain amino acids in the final product (vaccine) were in the following ranges: Tryptophan: 0.1-0.7 μM; Glutamine: 0.7-2.4 μM; Methionine: 0.5-3.2 μM; Arginine: 1.4-4.7 μM; Threonine: 1.6-8.0 μM; Lysine: 4.0-12.2 μM It was found / determined that.

[0101] In this regard, typical average concentrations of said amino acids in the final product (vaccine) are in the following ranges: Tryptophan: 0.2-0.6 μM; Glutamine: 0.8-2.2 μM; Methionine: 0.8-2.2 μM; Arginine: 1.6-4.3 μM; Threonine: 2.4-6.3 μM; Lysine: 4.4-11.2 μM It was also decided that. In conclusion, the results obtained from the analyses described in Examples 4-6 indicate that the method described in Example 1 provides products with certain unique properties, each of which may contribute to useful results of the tests and studies described in Examples 2 and 3.

[0102] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0103] [Figure 1] Figure 1 provides a schematic overview of an exemplary method according to the invention. SEQ ID NO: 1 corresponds to the sequence of the PCV2a ORF2 protein, and SEQ ID NO: 2 corresponds to the sequence of the PCV2b ORF2 protein, the "PCV2b ORF2 protein" referred to in this specification in particular relates to the ORF2 protein of the so-called "mutated PCV2b", which may also belong to genotype d.

[0104] This disclosure includes, inter alia, the following: 1. A method for producing an immunogenic composition comprising a recombinant protein, comprising: (a) - a first liquid, and - a recombinant protein and / or a quaternary structure comprising a plurality of said recombinant proteins providing a mixture containing (b) concentrating the recombinant protein and / or said quaternary structure in the mixture resulting from step (a) by removing a portion of the first liquid from the mixture; and (c) treating the solution resulting from step (b) by continuous diafiltration. The method includes: 2. The method of claim 1, wherein the continuous diafiltration is continuous diafiltration against a second liquid, the second liquid being different from the first liquid. 3. The mixture of step (a) - a vector comprising a nucleic acid sequence encoding said recombinant protein, said vector being inactivated by an inactivating agent, and / or - the inactivating agent has been neutralized by a neutralizing agent, and / or - Neutralizer 3. The method according to claim 1 or 2, further comprising: 4. The method according to item 3, wherein in step (c) the solution resulting from step (b) is treated by continuous diafiltration such that the concentration of neutralized inactivating agent and / or the concentration of neutralizing agent is reduced in the treated solution.

[0105] 5.(a)(i) - a first liquid; - a recombinant protein and / or a quaternary structure comprising a plurality of said recombinant proteins, - a vector comprising a nucleic acid sequence encoding said recombinant protein providing a mixture containing (ii) inactivating the vector by adding an inactivating agent to the mixture of step (i); (iii) neutralizing the deactivating agent by adding a neutralizing agent to the mixture resulting from step (ii); (b) concentrating the recombinant protein and / or said quaternary structure in the mixture resulting from step (a)(iii) by removing a portion of the first liquid from the mixture; and (c) treating the solution resulting from step (b) by continuous diafiltration such that the concentration of neutralized inactivating agent and / or the concentration of neutralizing agent is reduced in the treated solution. A method for producing an immunogenic composition comprising a recombinant protein, particularly the method according to any one of items 1 to 4. 6. The method according to any one of claims 1 to 5, wherein in step (b), the step of removing a portion of the first liquid from the mixture consists of or includes filtering the mixture through at least one filter, the at least one filter preferably comprising a filter membrane. 7. In step (b), the concentrating step comprises: - feeding the mixture into a filter system containing at least one filter, the at least one filter comprising a filter membrane having a membrane pore size that allows the passage of the neutralized inactivating agent and / or neutralizing agent while retaining the recombinant protein and / or said quaternary structure in the bulk stream; - Discharging the permeate containing the neutralized inactivating agent and / or neutralizing agent; The method according to any one of items 3 to 6, comprising:

[0106] 8. In step (c), the continuous diafiltration comprises: - feeding the solution into a filter system containing at least one filter, the at least one filter comprising a filter membrane having a membrane pore size that allows the passage of the neutralized inactivating agent and / or neutralizing agent while retaining the recombinant protein and / or said quaternary structure in the bulk stream; - Discharging the permeate containing the neutralized inactivating agent and / or neutralizing agent; - adding a second liquid to the bulk stream at a rate equal to the permeate stream, the second liquid being different from the first liquid; The method according to any one of items 3 to 7, comprising: 9. The method according to any one of items 6 to 8, wherein the at least one filter is at least one flat sheet filter and / or at least one hollow fiber filter, and the at least one flat sheet filter is preferably at least one cassette filter.

[0107] 10. At least one filter is 2 to 8 filters, preferably 5 to 7 filters; and / or at least one filter, each of which is approximately 16-26 m 2 , preferably about 20 to 22 m 2 having a total filter area of The method according to any one of items 6 to 9. 11. The method according to any one of items 6 to 10, wherein the filter membrane has an average pore size smaller than the recombinant protein and / or the quaternary structure, and / or the filter membrane has a molecular weight cut-off of between about 200 kDa and about 500 kDa. 12. The filter membrane is made of or comprises a material selected from the group consisting of polyethersulfone, cellulose hydrate, regenerated cellulose, stabilized cellulose, crosslinked cellulose, crosslinked cellulose hydrate, cellulose acetate, polyamide, polyurethane, polypropylene, polysulfone, polycarbonate, nylon, polyimide, and combinations thereof; and / or the filter membrane preferably consists of or comprises polyethersulfone, or the filter membrane may be a stabilized cellulose-based membrane, The method according to any one of items 6 to 11.

[0108] 13. The method according to any one of paragraphs 7 to 12, wherein the same filter system is used in step (b) and in step (c). 14. - step (iii) is carried out in a first container, the mixture resulting from the step of neutralizing the deactivating agent is transferred from the first container to a second container connected to a filter system, after transferring the mixture from the first container to the second container, the valve between the first container and the second container is closed, and the empty first container is filled with the second liquid, - in step (b), the mixture is circulated through the second vessel and the filter system until concentration is complete; and - in step (c), while the mixture is circulating through the filter system and the second container, a valve between the first and second containers is opened and a second liquid is continuously introduced from the first container to the second container, The method according to any one of items 5 to 13. 15. The method according to any one of items 1 to 14, wherein the first liquid comprises a portion of a cell culture medium or consists of a cell culture medium, and the cell culture medium is preferably an insect cell culture medium.

[0109] 16. The method according to any one of items 1 to 15, wherein the volume of the mixture obtained from step (a) is 1000L to 10000L, preferably 2000L to 8000L, and most preferably 3000L to 5000L. 17. The method according to any one of items 1 to 15, wherein in step (b), the recombinant protein and / or the quaternary structure is finally concentrated at least 5-fold, preferably at least 8-fold, more preferably 9-fold to 11-fold, compared to the volume of the mixture obtained from step (a). 18. The method according to any one of items 2 to 17, wherein the second liquid is a buffer solution, preferably P-saline or phosphate buffered saline (PBS). 19. The method according to any one of paragraphs 8 to 18, wherein in step (c), the total volume of the second liquid added to the bulk stream is at least 5 times, preferably at least 7 times, more preferably at least 9 times the volume of the solution resulting from step (b), and / or the total volume of the second liquid added to the bulk stream is most preferably about the same as the volume of the mixture resulting from step (a).

[0110] 20. (d) mixing the mixture remaining after step (c) with a further component selected from the group consisting of a pharma- ceutically acceptable carrier, adjuvant, diluent, excipient and combinations thereof, wherein the concentration of recombinant protein and / or the concentration of said quaternary structure in the solution resulting from said mixing step is preferably approximately the same as the concentration of recombinant protein and / or said quaternary structure in the mixture resulting from step (a). 20. The method according to any one of items 1 to 19, further comprising: 21. The mixture used for step (a) (1) allowing infection of susceptible cells in culture with a vector comprising a nucleic acid sequence encoding said recombinant protein, said recombinant protein being expressed by said vector; (2) subsequently recovering the recombinant protein and / or quaternary structures comprising said recombinant proteins from the cell culture, preferably in which the cell debris is separated from the recombinant protein and / or said quaternary structures by a separation step, preferably comprising microfiltration through at least one filter, preferably two filters, the at least one filter preferably having a pore size larger than the recombinant protein and / or the quaternary structures comprising said recombinant proteins, in particular having a pore size of about 1 to about 20 μm and / or about 0.1 μm to about 4 μm. 21. The method according to any one of items 1 to 20, which is obtained by a procedure comprising the steps of:

[0111] 22. The separation step comprises: - microfiltration through one or more filters having a pore size of about 2 μm to about 15 μm, and / or - Microfiltration through one or more filters with pore sizes of about 0.8 μm to about 1.0 μm 22. The method of claim 21, comprising or consisting of: 23. The method according to item 21 or 22, wherein the cell culture in step (1) is preferably maintained at 22 to 32°C during which the recombinant protein is expressed by the vector, and / or the harvesting in step (2) occurs 5 to 8 days, preferably 8 days, after inoculation of the cells with the vector. 24. The method according to any one of clauses 1 to 23, wherein the recombinant protein is a PCV2 ORF2 protein, preferably comprising or consisting of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.5% or 100% sequence identity to the sequence of SEQ ID NO:1 and / or SEQ ID NO:2.

[0112] 25. The method according to any one of paragraphs 1 to 24, wherein the quaternary structure is a virus-like particle. 26. The method of any one of clauses 3 to 25, wherein the vector is a recombinant virus, preferably a baculovirus, and / or the nucleic acid sequence is a DNA sequence. 27. The method of any one of paragraphs 3 to 26, wherein the inactivating agent is an aziridine compound, preferably binary ethyleneimine (BEI), and / or the inactivating agent is added in molar excess relative to the vector. 28. The method of any one of paragraphs 3 to 27, wherein the neutralizing agent is sodium thiosulfate and / or the neutralizing agent is added in molar excess relative to the inactivating agent. 29. The method according to any one of paragraphs 5 to 28, wherein in step (iii), the neutralizing agent is added in an equivalent amount compared to the amount of the inactivating agent added in step (ii). 30. The method according to any one of paragraphs 5 to 29, wherein the mixture remaining after step (c) contains an inactivating agent at a concentration less than 1000 times the concentration of the neutralizing agent obtained from step (iii).

[0113] 31. The virucidal activity of the immunogenic composition resulting from the method is reduced by at least 20% compared to a mixture of immunogenic compositions that has not been subjected to the concentration of step (b) and the continuous diafiltration of step (c) of the method, and / or the immunogenic composition produced by the method has a virucidal activity of at least 1 log TCID 35 per mL of live virus when the live virus is mixed with the immunogenic composition for 4 hours or more, particularly at room temperature. 50 Loss of less than 0.7 log TCID per mL of live virus, preferably 50 The method according to any one of items 1 to 30, wherein the method causes a loss of less than 10%. 32. The method of any one of paragraphs 1 to 31, further comprising mixing the mixture remaining after step (c) and / or step (d) with at least one additional antigen. 33. The method of clause 31 or 32, wherein the live virus or at least one additional antigen is a porcine reproductive and respiratory syndrome (PRRS) virus.

[0114] 34. An immunogenic composition obtainable by the method according to any one of items 1 to 33. 35. The immunogenic composition according to claim 34, which can be obtained by the method according to any one of claims 28 to 33, and wherein the mixture remaining after step (c) contains less than 3 μM sodium thiosulfate. 36. The immunogenic composition according to claim 34, which can be obtained by the method according to any one of claims 28 to 33, and the immunogenic composition obtained from step (d) is substantially free of sodium thiosulfate. 37. - A recombinant PCV2 ORF2 protein, which preferably comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.5% or 100% sequence identity with the sequences SEQ ID NO: 1 and / or SEQ ID NO: 2, as well as - 1-10 μM L-arginine and / or 1-10 μM L-lysine Including, Preferably, the immunogenic composition is substantially free of neutralized inactivating agents and / or substantially free of neutralizing agents, in particular the immunogenic composition according to any one of items 34 to 36.

[0115] 38. A recombinant PCV2 ORF2 protein, preferably comprising or consisting of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.5% or 100% sequence identity with the sequences of SEQ ID NO:1 and / or SEQ ID NO:2; And 0.1–1 μM L-tryptophan; and / or 0.1–3 μM L-glutamine; and / or 0.2-4 μM L-methionine; and / or 1-10 μM L-arginine; and / or 1-10 μM L-threonine; and / or 1-15 μM L-lysine An immunogenic composition comprising the immunogenic composition of any one of clauses 34 to 37, preferably an immunogenic composition obtained by any such method comprising step (d).

[0116] 39. 0.1 to 0.7 μM L-tryptophan; and / or 0.7–2.4 μM L-glutamine; and / or 0.5 to 3.2 µM L-methionine; and / or 1.4 to 4.7 μM L-arginine; and / or 1.6 to 8.0 µM L-threonine; and / or 4.0-12.2 μM L-lysine The immunogenic composition according to any one of items 34 to 38, comprising: 40. 0.2 to 0.6 μM L-tryptophan; and / or 0.8–2.2 μM L-glutamine; and / or 0.8 to 2.2 µM L-methionine; and / or 1.6 to 4.3 μM L-arginine; and / or 2.4 to 6.3 µM L-threonine; and / or 4.4 to 11.2 μM L-lysine The immunogenic composition of claim 39, comprising:

[0117] 41. Contains 0.7 to 2.4 μM L-glutamine and 4.0 to 12.2 μM L-lysine. The immunogenic composition according to any one of items 34 to 40. 42. Contains 0.8 to 2.2 μM L-glutamine and 4.4 to 11.2 μM L-lysine. Item 42. The immunogenic composition according to any one of Items 34 to 41. 43. The immunogenic composition, when subjected to two-dimensional (2D) ultra-performance liquid chromatography (UPLC) at room temperature, exhibits a ratio of the area of ​​peak A to the area of ​​peak B of 0.2 to 0.4, and / or a ratio of the area of ​​peak C to the area of ​​peak B of 0.5 to 0.9, and the 2D UPLC is A first dimension chromatography having a system volume of 400 μL, - a sample containing an immunogenic composition is injected into an anion exchange column having a column volume of 0.1 mL, the column being packed with a quaternary amine-functionalized material suitable for binding virus-like particles; and - a multi-step gradient method starting with 100% 50mM Tris, pH 8 at 0 min, 97.5% 50mM Tris, pH 8 and 2.5% 50mM Tris, 2M NaCl, pH 8 at 1 min, and finally 10% 50mM Tris, pH 8 and 90% 50mM Tris, 2M NaCl, pH 8 at 2 min through an anion exchange column at a flow rate of 0.6mL / min; A first dimension of chromatography is included, At a retention time of 2.67 minutes, a switch was made from the first dimension chromatography to the second dimension chromatography, and 50 μL of the eluate of the first dimension chromatography was passed through a size exclusion column having a column volume of 1.75 mL with a pore size of 450 Å at a flow rate of 0.3 mL / min; Chromatograms were recorded by monitoring the fluorescence emission at a wavelength of 330 nm and excitation at a wavelength of 280 nm; The area of ​​peak A is the peak area of ​​the highest peak in the chromatogram with a retention time of 0 to 1 minute, The area of ​​peak B is the peak area of ​​the highest peak at a retention time of 7 to 8 minutes in the chromatogram, The area of ​​peak C is the peak area of ​​the highest peak with a retention time of 8.5 to 9.5 minutes in the chromatogram. An immunogenic composition, in particular an immunogenic composition according to any one of clauses 34 to 42, preferably an immunogenic composition obtained by any such method comprising step (d).

[0118] 44. The immunogenic composition according to any one of clauses 34 to 43, which is substantially free of a neutralizing agent, preferably substantially free of sodium thiosulfate. 45. The immunogenic composition of any one of clauses 34 to 44, further comprising a live attenuated virus, preferably an attenuated porcine reproductive and respiratory syndrome (PRRS) virus, or a live attenuated bacterium. 46. ​​The immunogenic composition of any one of clauses 34 to 45, which induces a protective immune response against the pathogen from which the amino acid sequence of the recombinant protein is derived, preferably PCV2, after administration of one dose of the immunogenic composition.

[0119] 47. The immunogenic composition of clause 45 or 46, which induces a protective immune response against the PRRS virus following administration of one dose of the immunogenic composition. 48. A kit comprising a container containing the immunogenic composition according to any one of items 34 to 47. 49. A kit comprising a container containing an immunogenic composition produced by the method according to any one of items 1 to 33. 50. The kit of clause 48 or 49, further comprising at least one additional container containing at least one additional antigen selected from the group consisting of a live attenuated virus, preferably an attenuated PRRS virus, and a live attenuated bacterium.

[0120] 51. The immunogenic composition of any one of clauses 34-50, and / or the kit of any one of clauses 48-50, further comprising one or more attenuated or inactivated non-PRRSV pathogens, or antigenic material thereof. 52. The immunogenic composition according to any one of clauses 34 to 47 and 51, and / or the kit according to any one of clauses 48 to 50, for use as a medicament, preferably as a vaccine. 53. In animals: - induce a protective immune response against at least one pathogen, and / or - reducing one or more clinical signs of at least one pathogen infection; The method includes the steps of: The immunogenic composition according to any one of clauses 34 to 47 and 51 to 52, and / or the kit according to any one of clauses 48 to 52, wherein at least one pathogen is preferably selected from the group consisting of PCV2, PRRSV, a non-PRRSV pathogen, and combinations thereof.

[0121] 54. The immunogenic composition and / or kit according to clause 51, particularly for the use according to clause 52 or 53, wherein the non-PRRSV pathogen is selected from Mycoplasma hyopneumoniae, pseudorabies virus, swine influenza virus, porcine parvovirus, transmissible gastroenteritis virus, Escherichia coli, Erysipelothrix rhusiopathiae, Bordetella bronchiseptica, Salmonella cholerae suis, Haemophilus parasuis, Pasteurella multocida, Streptococcus suis and Actinobacillus pleuropneumoniae. 55. A method for producing an immunogenic composition comprising a recombinant protein, comprising: (a) - a first liquid, and - recombinant PCV2 ORF2 protein and / or virus-like particles containing multiple recombinant PCV2 ORF2 proteins providing a mixture containing (b) concentrating the recombinant PCV2 ORF2 protein and / or the virus-like particles in the mixture obtained from step (a) by removing a portion of the first liquid from the mixture to form a bulk flow solution; and (c) treating the bulk stream solution resulting from step (b) by continuous diafiltration to form a treated solution. The method includes:

[0122] 56. The mixture of step (a) - a vector comprising a nucleic acid sequence encoding the recombinant PCV2 ORF2 protein, the vector being inactivated by an inactivating agent, and / or - the inactivating agent has been neutralized by a neutralizing agent, and / or - Neutralizer and and / or in step (c), the bulk flow solution resulting from step (b) is treated by continuous diafiltration such that the concentration of neutralized inactivating agent and / or the concentration of neutralizing agent is reduced in the treated solution; Item 56. The method according to item 55.

[0123] 57.(a)(i) - First liquid, - a recombinant PCV2 ORF2 protein and / or a virus-like particle comprising a plurality of recombinant PCV2 ORF2 proteins, - a vector comprising a nucleic acid sequence encoding said recombinant protein providing a mixture containing (ii) inactivating the vector by adding an inactivating agent to the mixture of step (i); (iii) neutralizing the deactivating agent by adding a neutralizing agent to the mixture resulting from step (ii); (b) concentrating the recombinant PCV2 ORF2 protein and / or the virus-like particles in the mixture resulting from step (a)(iii) by removing a portion of the first liquid from the mixture to form a bulk flow solution; and (c) treating the bulk stream solution resulting from step (b) by continuous diafiltration such that the concentration of neutralized inactivating agent and / or the concentration of neutralizing agent is reduced in the treated solution to form a treated solution. 57. The method of claim 55 or 56, comprising:

[0124] 58. The method of any one of clauses 55 to 57, wherein in step (b), the step of removing a portion of the first liquid from the mixture consists of or includes filtering the mixture through at least one filter, the at least one filter preferably including a filter membrane.

[0125] 59. The mixture of step (a) - forming a permeate by feeding the mixture to a filter system containing at least one filter, the at least one filter comprising a filter membrane having a membrane pore size that allows the passage of the neutralized inactivation agent and / or neutralizing agent while retaining the recombinant PCV2 ORF2 protein and / or said virus-like particles in the bulk flow solution; - the permeate comprises a neutralized inactivating agent and / or a neutralizing agent; - Discharge of permeate is concentrated by and / or wherein the continuous diafiltration of step (c) comprises: - forming a permeate by feeding the bulk flow solution of step (b) to a filter system containing at least one filter, the at least one filter comprising a filter membrane having a membrane pore size that allows the passage of the neutralized inactivating agent and / or neutralizing agent while retaining the recombinant PCV2 ORF2 protein and / or said virus-like particles in the bulk flow solution; - the permeate comprises a neutralized inactivating agent and / or a neutralizing agent; - draining the permeate, and - adding a second liquid to the bulk flow solution at a rate equal to the rate at which the permeate is discharged; Including, The second liquid is different from the first liquid; 59. The method according to any one of items 55 to 58.

[0126] 60. The at least one filter is at least one flat sheet filter and / or at least one hollow fiber filter, the at least one flat sheet filter being preferably at least one cassette filter; and / or at least one filter is 2-8 filters, preferably 5-7 filters; and / or at least one filter each having a length of about 16-26 m 2 , preferably about 20 to 22 m 2 having a total filter area of and / or the filter membrane has an average pore size smaller than the recombinant PCV2 ORF2 protein and / or the virus-like particle, and / or the filter membrane has a molecular weight cut-off of between about 200 kDa and about 500 kDa; and / or the filter membrane is made of or comprises a material selected from the group consisting of polyethersulfone, cellulose hydrate, regenerated cellulose, stabilized cellulose, crosslinked cellulose, crosslinked cellulose hydrate, cellulose acetate, polyamide, polyurethane, polypropylene, polysulfone, polycarbonate, nylon, polyimide, and combinations thereof; and / or the filter membrane preferably consists of or comprises polyethersulfone, or the filter membrane may be a stabilized cellulose-based membrane, 60. The method according to item 58 or 59. 61. The method of claim 59 or 60, wherein the same filter system is utilized in step (b) and in step (c).

[0127] 62. - Step (iii) is carried out in a first container, the mixture of step (a) resulting from the step of neutralizing the deactivating agent is transferred from the first container to a second container connected to a filter system, after transferring the mixture of step (a) from the first container to the second container, the valve between the first container and the second container is closed and the empty first container is filled with the second liquid, - in step (b), the mixture of step (a) is circulated through the second vessel and the filter system until the mixture of step (a) is concentrated, thereby forming a bulk stream solution of step (b); and - in step (c), while the bulk flow solution is circulating through the filter system and the second vessel, a valve between the first and second vessels is opened and a second liquid is continuously introduced from the first vessel to the second vessel until a treatment solution is formed; Item 62. The method according to any one of items 57 to 61.

[0128] 63. The first liquid comprises a portion of or consists of a cell culture medium, the cell culture medium being preferably an insect cell culture medium; and / or the mixture resulting from step (a) has a volume, the volume of the mixture being between 1000 L and 10000 L, preferably between 2000 L and 8000 L, most preferably between 3000 L and 5000 L; 63. The method according to any one of claims 55 to 62, wherein in step (b), the mixture of step (a) is concentrated in the bulk flow solution until the recombinant PCV2 ORF2 protein and / or virus-like particles in the bulk flow solution are concentrated at least 5-fold, preferably at least 8-fold, more preferably 9-fold to 11-fold, compared to the concentration of the recombinant PCV2 ORF2 protein and / or virus-like particles in the volume of the mixture obtained from step (a). 64. The second liquid is a buffer solution, preferably P-saline or phosphate buffered saline (PBS); 64. The method of any one of claims 59 to 63, wherein in step (c) the total volume of the second liquid added to the bulk stream solution is at least 5 times, preferably at least 7 times, more preferably at least 9 times the volume of the bulk stream solution resulting from step (b), and / or the total volume of the second liquid added to the bulk stream solution is most preferably about the same as the volume of the mixture resulting from concentrated step (a).

[0129] 65. (d) Forming solution D by mixing the treatment solution remaining after step (c) with additional components selected from the group consisting of pharma- ceutically acceptable carriers, adjuvants, diluents, excipients, and combinations thereof; the concentration of recombinant PCV2 ORF2 protein and / or the concentration of said virus-like particles in solution D resulting from said mixing step is preferably approximately the same as the concentration of PCV2 ORF2 recombinant protein and / or the virus-like particles in the mixture resulting from step (a); Item 65. The method according to any one of items 55 to 64, further comprising: 66. The method according to any one of clauses 55 to 65, wherein the recombinant PCV2 ORF2 protein comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.5% or 100% sequence identity to the sequence of SEQ ID NO:1 and / or SEQ ID NO:2. 67. The vector is a recombinant virus, preferably a baculovirus, and / or the nucleic acid sequence is a DNA sequence; and / or the inactivating agent is an aziridine compound, preferably binary ethyleneimine (BEI), and / or the inactivating agent is added in molar excess relative to the vector; 67. The method according to any one of items 56 to 66, wherein the neutralizing agent is sodium thiosulfate and / or the neutralizing agent is added in molar excess relative to the inactivating agent.

[0130] 68. The immunogenic composition in solution D has virucidal activity, and the virucidal activity of the immunogenic composition obtained from said process is reduced by at least 20% compared to an immunogenic composition not produced by the concentration of step (b) and the continuous diafiltration of step (c) of said process, and / or the immunogenic composition produced by said process has a virucidal activity of at least 1 log TCID 3 per mL of live virus when the live virus is mixed with the immunogenic composition for 4 hours or more. 50 Loss of less than 0.7 log TCID per mL of live virus, preferably 50 68. The method according to any one of claims 55 to 67, wherein the method causes a loss of less than 100%. 69. The method of clause 68, wherein the live virus is mixed with the immunogenic composition at room temperature for more than 4 hours.

[0131] 70. The method further comprises the step of mixing the treatment solution remaining after step (c) and / or solution D remaining after step (d) with at least one additional antigen; and / or wherein at least one additional antigen is preferably the porcine reproductive and respiratory syndrome (PRRS) virus; Item 70. The method according to any one of items 55 to 69. 71. An immunogenic composition produced by the method according to any one of clauses 55 to 70. 72. - A recombinant PCV2 ORF2 protein, which preferably comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.5% or 100% sequence identity with the sequences SEQ ID NO: 1 and / or SEQ ID NO: 2, as well as - 1-10 μM L-arginine and / or 1-10 μM L-lysine Including, Preferably, substantially free of neutralized deactivating agents and / or substantially free of neutralizing agents; Item 72. The immunogenic composition of item 71.

[0132] 73. The method further comprises the steps of: (a) detecting a live attenuated virus, preferably an attenuated porcine reproductive and respiratory syndrome (PRRS) virus, or an attenuated live bacterium; and / or induce a protective immune response against PCV2 after administration of one dose of the immunogenic composition; and / or induce a protective immune response against PRRS virus following administration of one dose of the immunogenic composition; Item 73. The immunogenic composition of item 71 or 72. 74. Preferably: At least one additional container containing at least one additional antigen selected from the group consisting of an attenuated live virus, preferably an attenuated PRRS virus, and an attenuated live bacterium; A kit comprising a container containing the immunogenic composition according to any one of items 71 to 73. 75. The immunogenic composition of any one of clauses 71 to 73, and / or the kit of clause 74, for use as a medicament, preferably as a vaccine.

[0133] 76. In animals: - induce a protective immune response against at least one pathogen, and / or - reducing one or more clinical signs of at least one pathogen infection; The method includes the steps of: The immunogenic composition of any one of clauses 71 to 73 and 75, and / or the kit of clause 74 or 75, wherein at least one pathogen is preferably selected from the group consisting of PCV2, PRRSV, a non-PRRSV pathogen, and combinations thereof.

Claims

1. 1. A method for producing an immunogenic composition comprising a recombinant protein, comprising: (a) a first liquid, and - recombinant PCV2 ORF2 protein and / or virus-like particles containing multiple recombinant PCV2 ORF2 proteins providing a mixture containing (b) concentrating the recombinant PCV2 ORF2 protein and / or the virus-like particles in the mixture obtained from step (a) by removing a portion of the first liquid from the mixture; and (c) treating the solution resulting from step (b) by continuous diafiltration. The method includes:

2. The mixture of step (a) - a vector comprising a nucleic acid sequence encoding said recombinant PCV2 ORF2 protein, said vector being inactivated by an inactivating agent, and / or - the inactivating agent has been neutralized by a neutralizing agent, and / or - Neutralizer and and / or in step (c), the solution resulting from step (b) is treated by continuous diafiltration such that the concentration of neutralized inactivating agent and / or the concentration of neutralizing agent is reduced in the treated solution; The method of claim 1.

3. (a)(i)—a first liquid; - a virus-like particle comprising a recombinant PCV2 ORF2 protein and / or a plurality of recombinant PCV2 ORF2 proteins, and - a vector comprising a nucleic acid sequence encoding said recombinant protein providing a mixture containing (ii) inactivating the vector by adding an inactivating agent to the mixture of step (i); (iii) neutralizing the deactivating agent by adding a neutralizing agent to the mixture resulting from step (ii); (b) concentrating the recombinant PCV2 ORF2 protein and / or the virus-like particles in the mixture resulting from step (a)(iii) by removing a portion of the first liquid from the mixture; and (c) treating the solution resulting from step (b) by continuous diafiltration such that the concentration of neutralized deactivating agent and / or the concentration of neutralizing agent is reduced in the treated solution.

2. A method for producing an immunogenic composition comprising a recombinant PCV2 ORF2 protein, in particular the method according to claim 2, comprising:

4. 4. The method according to any one of claims 1 to 3, wherein in step (b), removing a portion of the first liquid from the mixture consists of or comprises filtering the mixture through at least one filter, said at least one filter preferably comprising a filter membrane.

5. In step (b), the concentrating step comprises: - feeding the mixture into a filter system containing at least one filter, wherein the at least one filter comprises a filter membrane having a membrane pore size that allows the passage of the neutralized inactivating agent and / or neutralizing agent while retaining the recombinant PCV2 ORF2 protein and / or said virus-like particles in the bulk stream; - Discharging the permeate containing the neutralized deactivating agent and / or neutralizing agent. Including, and / or wherein in step (c), the continuous diafiltration comprises: - feeding the solution into a filter system containing at least one filter, wherein the at least one filter comprises a filter membrane having a membrane pore size that allows the passage of the neutralized inactivating agent and / or neutralizing agent while retaining the recombinant PCV2 ORF2 protein and / or said virus-like particles in the bulk stream; - Discharging the permeate containing the neutralized deactivating agent and / or neutralizing agent, - adding a second liquid to the bulk stream at a rate equal to the permeate stream, where the second liquid is different from the first liquid; The method according to any one of claims 2 to 4, comprising:

6. the at least one filter is at least one flat sheet filter and / or at least one hollow fiber filter, the at least one flat sheet filter being preferably at least one cassette filter; and / or at least one filter is 2-8 filters, preferably 5-7 filters; and / or each of the at least one filter is about 16 to 26 m 2 , preferably about 20 to 22 m 2 having a total filter area of and / or the filter membrane has an average pore size smaller than the recombinant PCV2 ORF2 protein and / or said virus-like particle, and / or the filter membrane has a molecular weight cut-off of between about 200 kDa and about 500 kDa; and / or the filter membrane is made of or comprises a material selected from the group consisting of polyethersulfone, cellulose hydrate, regenerated cellulose, stabilized cellulose, crosslinked cellulose, crosslinked cellulose hydrate, cellulose acetate, polyamide, polyurethane, polypropylene, polysulfone, polycarbonate, nylon, polyimide, and combinations thereof; 6. The method according to claim 4 or 5, wherein the filter membrane preferably consists of or comprises polyethersulfone, or the filter membrane may be a stabilized cellulose-based membrane.

7. 7. The method of claim 5 or 6, wherein in step (b) and in step (c) the same filter system is utilized.

8. - step (iii) is carried out in a first container, the mixture resulting from the step of neutralizing the deactivating agent is transferred from the first container to a second container connected to a filter system, after transferring the mixture from the first container to the second container the valve between the first container and the second container is closed and the empty first container is filled with the second liquid, - in step (b), the mixture is circulated through the second vessel and the filter system until concentration is complete; - in step (c), while the mixture is circulating through the filter system and the second container, a valve between the first and second containers is opened and a second liquid is continuously introduced from the first container to the second container; The method according to any one of claims 3 to 7.

9. the first liquid comprises a portion of or consists of a cell culture medium, the cell culture medium being preferably an insect cell culture medium; and / or the volume of the mixture resulting from step (a) is between 1000L and 10000L, preferably between 2000L and 8000L, most preferably between 3000L and 5000L; and / or wherein in step (b), the recombinant PCV2 ORF2 protein and / or the virus-like particles are finally concentrated at least 5-fold, preferably at least 8-fold, more preferably 9-fold to 11-fold, compared to the volume of the mixture obtained from step (a).

10. the second liquid is a buffer solution, preferably P-saline or phosphate buffered saline (PBS); and / or wherein in step (c) the total volume of the second liquid added to the bulk stream is at least 5 times, preferably at least 7 times, more preferably at least 9 times the volume of the solution resulting from step (b); and / or the total volume of the second liquid added to the bulk stream is most preferably about the same as the volume of the mixture resulting from step (a).

11. (d) mixing the mixture remaining after step (c) with a further component selected from the group consisting of a pharma- ceutically acceptable carrier, an adjuvant, a diluent, an excipient, and combinations thereof, wherein the concentration of recombinant PCV2 ORF2 protein and / or the concentration of said virus-like particle structures in the solution resulting from said mixing step is preferably about the same as the concentration of PCV2 ORF2 recombinant protein and / or said virus-like particles in the mixture resulting from step (a). The method of any one of claims 1 to 10, further comprising:

12. The method of any one of claims 1 to 11, wherein the recombinant PCV2 ORF2 protein comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.5% or 100% sequence identity to the sequences of SEQ ID NO:1 and / or SEQ ID NO:

2.

13. the vector is a recombinant virus, preferably a baculovirus, and / or the nucleic acid sequence is a DNA sequence, and / or the inactivating agent is an aziridine compound, preferably binary ethyleneimine (BEI), and / or the inactivating agent is added in molar excess relative to the vector; and / or the neutralizing agent is sodium thiosulfate and / or the neutralizing agent is added in molar excess relative to the inactivating agent; The method according to any one of claims 2 to 12.

14. The virucidal activity of the immunogenic composition resulting from said process is reduced by at least 20% compared to a mixture of immunogenic compositions that has not been subjected to the concentration of step (b) and the continuous diafiltration of step (c) of said process, and / or the immunogenic composition produced by said process has a virucidal activity of at least 1 log TCID 3 per mL of live virus when the live virus is mixed with the immunogenic composition for 4 hours or more, in particular at room temperature. 50 Loss of less than 0.7 log TCID per mL of live virus 50 The method of any one of claims 1 to 13, wherein the method causes a loss of less than

15. further comprising the step of mixing the mixture remaining after step (c) and / or step (d) with at least one additional antigen; and / or wherein at least one additional antigen is preferably the porcine reproductive and respiratory syndrome (PRRS) virus; The method according to any one of claims 1 to 14.

16. An immunogenic composition obtainable by the method according to any one of claims 1 to 15.

17. - a recombinant PCV2 ORF2 protein, which preferably comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.5% or 100% sequence identity with the sequences SEQ ID NO: 1 and / or SEQ ID NO: 2, and 1-10 μM L-arginine and / or 1-10 μM L-lysine Including, Preferably, substantially free of neutralized deactivating agents and / or substantially free of neutralizing agents; An immunogenic composition, in particular an immunogenic composition according to claim 16.

18. further comprising a live attenuated virus, preferably an attenuated porcine reproductive and respiratory syndrome (PRRS) virus, or a live attenuated bacterium, and / or induce a protective immune response against PCV2 after administration of one dose of the immunogenic composition; and / or induce a protective immune response against the PRRS virus following administration of a single dose of the immunogenic composition; 18. The immunogenic composition of claim 16 or 17.

19. A kit comprising a container containing the immunogenic composition according to any one of claims 16 to 18, preferably comprising: At least one additional container containing at least one additional antigen selected from the group consisting of a live attenuated virus, preferably an attenuated PRRS virus, and a live attenuated bacterium. kit.

20. An immunogenic composition according to any one of claims 16 to 18 and / or a kit according to claim 19 for use as a medicament, preferably as a vaccine.

21. In animals, - induce a protective immune response against at least one pathogen, and / or - reducing one or more clinical signs of at least one pathogen infection, The method includes the steps of: The at least one pathogen is preferably selected from the group consisting of PCV2, PRRSV, a non-PRRSV pathogen, and combinations thereof; An immunogenic composition according to any one of claims 16 to 18 and 20 and / or a kit according to claim 19 or 20.

Images