Method for preparing immunogenic composition

Abstract

The present invention relates to a method of preparing an immunogenic composition exhibiting reduced viral activity, the method comprising the steps of: (a) providing a mixture having 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) treating the solution produced from step (b) by continuous diafiltration. The present invention enables the preparation of immunogenic compositions comprising recombinant proteins with reduced viral effects in a larger scale and even in a very large scale.

Description

Technical Field

[0001] The present invention relates to methods of preparing immunogenic compositions and to such immunogenic compositions that exhibit reduced virucidal activity. Background Art

[0002] Porcine circovirus type 2 (PCV2) is a small (17-22 nm in diameter), icosahedral, non-enveloped DNA virus containing 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-virulent, pigs infected with PCV2 exhibit a syndrome commonly referred to as post-weaning multisystemic wasting syndrome (PMWS). The clinical features of PMWS are emaciation, pale skin, unthriftiness, respiratory distress, diarrhea, icterus, and jaundice. In some affected pigs, a combination of all symptoms will be evident, while other pigs will have only one or two of these symptoms. During necropsy, microscopic and macroscopic lesions also appear on multiple tissues and organs, with lymphoid organs being the most common sites of lesions. A strong correlation has been observed between the amount of PCV2 nucleic acid or antigen and the severity of microscopic lymphoid lesions. The mortality rate of pigs infected with PCV2 can approach 80%. In addition to PMWS, PCV2 has been associated with several other infections including pseudorabies, porcine reproductive and respiratory syndrome (PRRS), Glasser's disease, streptococcal meningitis, salmonellosis, postweaning colibacillosis, dietary liver disease, and suppurative bronchopneumonia.

[0003] There are several vaccines available to reduce the effects of PCV2 infection in pigs. U.S. Pat. No. 6,703,023 provides a DNA-based vaccine for the prevention of PMWS in pigs. In WO 03 / 049703, the preparation of a live chimeric vaccine is described, which comprises a non-pathogenic PCV1 virus, however, wherein the ORF2 protein is replaced by the ORF2 protein of the pathogen PCV2. WO 99 / 18214 and WO 99 / 29717 have provided several PCV2 strains and methods for preparing killed PVC2 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. Another ORF2-based subunit vaccine is 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 an SDS-PAGE gel, has been used in the past as an antigenic component of vaccines and immunogenic compositions for PCV2. Typical methods for obtaining ORF2 for use in these vaccines and compositions generally consist of amplifying PCV2 DNA encoding ORF2, expressing the ORF2 protein in host cells, and extracting the ORF2 protein from the host cells via cell lysis. The recovered ORF2 cell lysate is then used as the antigenic portion of an immunogenic composition or vaccine. In some cases, the cell lysate containing ORF2 is separated from the cell debris.

[0005] Immunogenic compositions against PCV2 and various immunogenic compositions against other pathogens generally have a virucidal effect on 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 cannot result in a loss of more than 0.7 log / mL of live virus or less than 0.7 log / mL CFU of live bacteria when combined with the other components of the immunogenic composition. Compositions with greater virucidal activity than permitted cannot be combined with other antigens to produce a multivalent vaccine.

[0006] To this end, WO 11 / 28888 provides a method for reducing the virucidal activity of a composition comprising a PCV2 antigen, as well as an antigenic preparation and an immunogenic composition comprising a PCV2 antigen, wherein the virucidal activity has been reduced. More specifically, WO 11 / 28888 teaches a method for large-scale preparation of the PCV2 antigenic composition, wherein the method comprises the following steps:

[0007] i) obtaining a first liquid containing PCV2 antigens and virus-like particles containing ORF2 protein; and

[0008] ii) removing at least a portion of the first liquid from the PCV2 antigen of the virus-like particles comprising the ORF2 protein by a filtering step using a filter, wherein the filter comprises a semi-permeable membrane having an average pore size smaller than the PCV-2 antigen, thereby preventing at least 90% of the PCV2 antigen from passing through the semi-permeable membrane pores and retaining the PCV2 antigen within the filter, wherein the portion of the first liquid is removed from the PCV2 antigen by exchanging a portion of the first liquid with a second liquid, wherein the second liquid is different from the first liquid, and wherein the exchanging of the portion of the first liquid with the second liquid comprises the following steps:

[0009] a) adding a liquid, comprising adding a second liquid to a first liquid containing a PCV-2 antigen; and

[0010] b) concentrating the PCV-2 antigen to 3X to 50X the volume of the first liquid by removing a portion of the first and second liquids.

[0011] However, the liquid addition step a), comprising adding the second liquid preferably in excess volume to the first liquid, may lead to volume limitations with respect to the overall production scale, since the resulting volumes of the first and second liquids are typically increased multiple times compared to the volume of the first liquid before the concentration step b).

[0012] Therefore, other methods are desired to prepare immunogenic compositions comprising recombinant proteins with reduced virucidal effects still on a larger scale and even on an extremely large scale.

[0013] Description of the invention

[0014] The solution to the above technical problem is obtained by the description and embodiments characterized in the claims.

[0015] Hence, different aspects of the invention are realized according to the claims.

[0016] The present invention is based on the surprising finding that a method for preparing an immunogenic composition comprising a recombinant protein is sufficient to achieve the desired purpose, wherein the method preferably consists of or comprises the following steps in the following order:

[0017] (a) providing a mixture comprising:

[0018] - a first liquid, and

[0019] - a recombinant protein and / or a quaternary structure comprising a plurality of said recombinant proteins,

[0020] (b) concentrating the recombinant protein and / or the quaternary structure in the mixture produced from step (a) by removing a portion of the first liquid from the mixture, and

[0021] (c) treating the solution resulting from step (b) by continuous diafiltration, in particular by continuous diafiltration with a second liquid, wherein the second liquid is different from the first liquid.

[0022] It should be understood that the term "first liquid" as used herein is equivalent to the term "initial liquid" or "liquid (1)", respectively. Therefore, the term "first liquid" is interchangeable with the term "liquid (1)" or the term "initial liquid".

[0023] It is further understood that the term "second liquid" as used herein is equivalent to the term "further liquid" or "liquid (2)", respectively. Therefore, the term "second liquid" is interchangeable with the term "liquid (2)" or the term "further liquid".

[0024] In a preferred aspect, the mixture of step (a) further contains a vector comprising a nucleic acid sequence encoding the recombinant protein, wherein the vector has been inactivated by an inactivating agent. According to another preferred aspect, the mixture of step (a) further contains an inactivating agent, which has been neutralized by a neutralizing agent.

[0025] In yet another preferred aspect, the mixture of step (a) additionally contains a neutralizing agent.

[0026] As mentioned herein, it is particularly to be understood that the term "mixture of step (a)" is synonymous with "mixture provided in step (a)". As used herein, the term "provided in step" is particularly to be understood as being equivalent to "provided by step".

[0027] The present invention further relates to a process as described and / or claimed herein, wherein the mixture of step (a) above additionally comprises

[0028] - a vector comprising a nucleic acid sequence encoding the recombinant protein, wherein the vector has been inactivated by an inactivating agent, and / or

[0029] - an inactivating agent, which has been neutralized by a neutralizing agent, and / or

[0030] - neutralizing agent,

[0031] And preferably, the mixture of step (a) further contains the carrier, the neutralized inactivating agent and the neutralizing agent.

[0032] 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 in the treated solution is reduced.

[0033] Before describing aspects of the present invention, it must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to an "antigen" includes plural antigens, reference to a "virus" is a reference to one or more viruses known to those skilled in the art and their equivalents, and so forth. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods, apparatus, and materials are presently described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing cell lines, vectors, and methods as reported in publications that may be used in conjunction with the present invention. Nothing herein shall be construed as an admission that the present invention is not entitled to antedate such disclosures by virtue of prior invention.

[0034] In one aspect, the present invention relates to a method for preparing an immunogenic composition comprising a recombinant protein, wherein the method consists of or comprises the following steps, preferably in the following order:

[0035] (a)(i) Providing a mixture comprising:

[0036] - a first liquid,

[0037] - a recombinant protein and / or a quaternary structure comprising a plurality of said recombinant proteins, and

[0038] - a vector comprising a nucleic acid sequence encoding the recombinant protein;

[0039] (ii) inactivating the vector by adding an inactivating agent to the mixture of step (i);

[0040] (iii) neutralizing the inactivating agent by adding a neutralizing agent to the mixture resulting from step (ii);

[0041] (b) concentrating the recombinant protein and / or the quaternary structure in the mixture produced by step (a)(iii) by removing a portion of the first liquid from the mixture,

[0042] (c) and 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 in the treated solution is reduced.

[0043] In particular, the continuous diafiltration of step (c) is a continuous diafiltration with a second liquid, wherein the second liquid is different from the first liquid.

[0044] In the context of the present invention, it should be particularly understood that “(i)”, “(ii)” and “(iii)” respectively represent sub-steps of step (a), step (a) consists of or includes sub-steps “(i)”, “(ii)” and “(iii)”, and therefore, “(i)” is equivalent to “(a)(i)”, “(ii)” is equivalent to “(a)(ii)”, and “(iii)” is equivalent to “(a)(iii)”.

[0045] As mentioned herein, it is particularly to be understood that the terms "mixture of step (i)" and "mixture of step (a)(i)" are synonymous with "mixture provided in step (i)" and "mixture provided in step (a)(i)", respectively.

[0046] For the purposes of the present invention, "first liquid" refers to a liquid, aqueous or fluid medium that is usually 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 for preparing a recombinant protein in a cultured host cell. The cultured host cell may be a bacterium, yeast, insect cell, animal cell, and mammalian cell, with insect and mammalian cells being particularly preferred. Therefore, the first liquid may comprise or consist of a culture medium for culturing bacteria, yeast, insect cell, animal cell, or mammalian cell. Preferably, when insect cells are used, the cell culture medium is a serum-free cell culture medium, and most preferably, the culture medium is Excell 420 serum-free medium.

[0047] In another aspect, the present invention therefore relates to a method as described and claimed herein, wherein the first liquid comprises part of or consists of a cell culture medium, and wherein the cell culture medium is preferably an insect cell culture medium.

[0048] 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 can be anything less than 100% of the volume of the liquid (e.g., 90% of the liquid, 80% of the liquid, 70% of the liquid) and all amounts between more than 0% and less than 100%.

[0049] As used herein, the term "recombinant protein" refers in particular to proteins prepared by recombinant DNA technology, wherein DNA encoding the expressed protein is usually inserted into a suitable expression vector, which in turn is used to transform or, in the case of a viral vector, infect a host cell to produce a heterologous protein. Thus, as used herein, the term "recombinant protein" refers in particular to a protein molecule expressed from a recombinant DNA molecule. As used herein, a "recombinant DNA molecule" refers to a DNA molecule comprising DNA segments joined together by molecular biology techniques. Suitable systems for preparing recombinant proteins include, but are not limited to, insect cells (e.g., baculovirus), prokaryotic systems (e.g., Escherichia 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).

[0050] In the context of the present invention, the recombinant protein is preferably a recombinant PCV2 ORF2 protein.

[0051] The term "quaternary structure" as used in the context of the present invention particularly relates to the assembly of multiple folded protein molecules in a multi-subunit complex and particularly to virus-like particles.

[0052] 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 particularly to be understood that the term "plurality of said recombinant proteins" is equivalent to "plurality of recombinant proteins". It is particularly to be understood that said term means a virus-like particle comprising several molecules of a specific recombinant protein (e.g., recombinant PCV2 ORF2 protein). "Inactivated vector" in the context of the present invention particularly means that a vector that is normally capable of replication is rendered incapable of replication. "Inactivation" particularly refers to the process of inactivating a vector.

[0053] For the purposes of the present invention, "inactivating agent" refers to any substance 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), which includes a solution of 2-bromoethyleneamine hydrobromide (BEA) (which cyclizes to binary ethyleneimine (BEI)). Preferred other chemical inactivating agents include (but are not limited to) Triton X-100, sodium deoxycholate, cetyltrimethylammonium bromide, β-propiolactone, thimerosal, phenol and formaldehyde (Formalin). As used herein, the term "inactivating agent" therefore particularly relates to a chemical agent that is capable of modifying a vector by a chemical reaction, thereby rendering the vector incapable of replication.

[0054] 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 carrier, and in particular in molar excess relative to the nitrogenous bases of the carrier DNA base pairs reacting with BEI.

[0055] For the purposes of the present invention, "neutralizing agent" refers to any agent capable of neutralizing an inactivating agent as described herein such that the inactivating agent is no longer capable of inactivating the carrier. If an aziridine compound is used for inactivation, preferably, a nucleophile that opens a 3-membered ring is used for neutralization. The substance that neutralizes the inactivating agent is preferably sodium thiosulfate, sodium bisulfite, etc.

[0056] It is particularly preferred that the neutralizing agent is sodium thiosulfate and / or that the neutralizing agent is added in a molar excess relative to the inactivating agent.

[0057] As used herein, "neutralized inactivator" particularly relates to one or more products resulting from a chemical reaction of an inactivator with a neutralizing agent, such as the products of a chemical reaction of BEI with thiosulfate or other nucleophiles.

[0058] Any conventional chemical inactivation method can be used to inactivate the carrier. In a preferred form, for chemical treatment, the temperature is between about 32°C-42°C, more preferably between about 34°C-40°C, and most preferably between about 35°C-39°C. Preferred inactivation methods include adding cyclized binary ethyleneimine (BEI), preferably at a concentration of about 1 to about 20mM, preferably about 2 to about 10mM, still more preferably about 2 to about 8mM, still more preferably about 3 to about 7mM, most preferably about 5mM. For example, inactivation includes adding preferably about 0.4M 2-bromoethyleneamine hydrobromide (BEA) solution (which is cyclized to 0.2M binary ethyleneimine (BEI) in 0.3N NaOH) to the fluid, to produce a final concentration of about 5mM BEI. Preferably, the fluid is then stirred continuously for 2-96 hours. After inactivation is complete, sodium thiosulfate solution is preferably added at 1.0 M to neutralize any residual BEI, and the inactivated / neutralized harvest fluid can be stored frozen at -40°C or lower or between about 1°C-7°C. Preferably, an equivalent amount of sodium thiosulfate is added compared to the BEI added prior to inactivation. For example, if BEI is added to a final concentration of 5 mM, a 1.0 M sodium thiosulfate solution is added to produce a final minimum concentration of 5 mM to neutralize any residual BEI.

[0059] Thus, in step (iii), preferably an equivalent amount of neutralizing agent is added compared to the amount of inactivating agent added in step (ii). In the context of ranges, as described herein, it is specifically understood that "yz" is equivalent to "yz", and both are equivalent to "y to z", respectively, such that, for example, "0.1-1.0" is equivalent to "0.1-1.0".

[0060] For the purposes of the present invention, "vector" and "vector comprising a nucleic acid sequence encoding the recombinant protein" refer to a suitable expression vector, preferably a baculovirus expression vector, which is in turn used to transfect a host cell or, in the case of a baculovirus expression vector, infect a host cell to produce a protein or polypeptide encoded by the DNA. Vectors and methods for preparing and / or using vectors (or recombinants) for expression can be performed or accomplished, inter alia, by the methods disclosed in or analogously to the methods disclosed 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. Patent 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, pp. 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; EPA 0 370 573; U.S. Application No. 920,197 filed October 16, 1986; EP Patent Publication No. 265785; U.S. Patent No. 4,769,331 (recombinant herpes virus); 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. Pat. Nos. 5,591,439 and 5,552,143; WO 98 / 00166; granted U.S. application serial Nos. 08 / 675,556 and 08 / 675,566, both filed July 3, 1996 (recombinant adenovirus); Grunhaus et al., 1992, "Adenovirus as cloning vectors", Seminars in Virology (Vol. 3), pp. 237-52, 1993; Ballay et al., EMBO Journal, Vol. 4, pp. 3861-65, Graham, Tibtech 8, 85-87, April 1990; Prevec et al., J. Gen Virol. 70, 42434; PCT 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. Patents Nos. 5,591,639, 5,589,466 and 5,580,859, and WO 90 / 11092, WO 93 / 19183, WO 94 / 21797, WO 95 / 11307, WO 95 / 20660; Tang et al., Nature, and Furth et al., Analytical Biochemistry, which particularly relate to DNA expression vectors. See also WO 98 / 33510; Ju et al., Diabetologia, 41:736-739, 1998 (lentiviral expression system); Sanford et al., U.S. Pat. No. 4,945,050; Fischbach et al. (Intracel); WO 90 / 01543; Robinson et al., Seminars in Immunology, Vol. 9, pp. 271-283 (1997), (DNA vector system); 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); and other documents cited herein. .

[0061] Preferred viral vectors include baculovirus, such as BaculoGold (BD Biosciences Pharmingen, San Diego, CA), particularly provided in the case where the production cells are insect cells. Although baculovirus expression systems are preferred, it will be appreciated by those skilled in the art that other expression systems (including those described above) will work for the purposes of the present invention, i.e., expression of recombinant proteins.

[0062] 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.

[0063] Preferably, in step (b) of the method as described and claimed herein, removing a portion of the first liquid from the mixture consists of or comprises filtering the mixture through at least one filter, wherein the at least one filter preferably comprises a filter membrane.

[0064] According to another preferred aspect, in step (b), the concentration comprises

[0065] - feeding the mixture into a filter system comprising at least one filter, wherein the at least one filter comprises a filter membrane having a membrane pore size that allows the neutralized inactivating agent and / or neutralizing agent to pass therethrough while retaining the recombinant protein and / or the quaternary structure in the bulk flow, and

[0066] - Discharging the permeate comprising the neutralized inactivating agent and / or neutralizing agent.

[0067] According to another preferred aspect, in step (c), the continuous diafiltration comprises

[0068] - feeding the solution into a filter system comprising at least one filter, wherein the at least one filter comprises a filter membrane having a membrane pore size that allows the neutralized inactivating agent and / or neutralizing agent to pass therethrough while retaining the recombinant protein and / or the quaternary structure in the bulk flow, and

[0069] - discharging the permeate comprising the neutralized inactivating agent and / or neutralizing agent, and

[0070] - Adding a second liquid to the bulk flow at a rate equal to the permeate flow, wherein the second liquid is different from the first liquid.

[0071] For the purposes of the present invention, "second liquid" refers to any liquid that is typically used in combination with cells, antigens, immunogenic compositions, vaccines, etc., which is different from the first liquid. Preferably, the second liquid is an aqueous solution, even more preferably a pharmaceutically acceptable solution, and even more preferably a buffer, most preferably a physiologically acceptable buffer, such as saline or phosphate buffer, etc., such as P-saline or PBS. Most preferably, the second liquid is characterized in that it is non-virucidal to any live virus or live bacteria when the live virus or live bacteria are cultured or stored in the liquid.

[0072] Preferably, the at least one filter described herein is at least one flat plate filter and / or at least one hollow fiber filter, wherein the at least one filter is most preferably at least one flat plate filter. The at least one flat plate filter is preferably at least one cassette filter.

[0073] In particular, the at least one filter is 2-8 filters, preferably 5-7 filters, and most preferably 6 filters, and / or each of the at least one filter has in particular a diameter of about 16-26 m 2 , preferably about 20-22m 2 , most preferably about 21m 2 of total filter area.

[0074] The filter membrane as described herein preferably has an average pore size smaller than the recombinant protein and / or smaller than the quaternary structure, and / or the filter membrane preferably has a molecular weight cut-off between about 200 kDa and about 500 kDa, in particular about 300 kDa.

[0075] Preferably, the filter membrane consists of or comprises a material selected from the group consisting of polyethersulfone, cellulose hydrate, regenerated cellulose, stabilized cellulose, cross-linked cellulose, cross-linked cellulose hydrate, cellulose acetate, polyamide, polyurethane, polypropylene, polysulfone, polycarbonate, nylon, polyimide, and combinations thereof,

[0076] And / or the filter membrane preferably consists of or comprises polyethersulfone, or the filter membrane is optionally a stabilized cellulose-based membrane.

[0077] According to another preferred aspect of the present invention, in step (b) and in step (c), the same filter system is utilized. Thus, according to this preferred aspect, only one filter system is used for the concentration of step (b) and the continuous diafiltration of step (c).

[0078] Preferably, the mixture remaining after step (c) comprises an inactivating agent concentration that is less than one thousandth of the neutralising agent concentration resulting from step (iii).

[0079] Furthermore, in the context of the present invention, it has been found that, particularly preferably,

[0080] - step (iii) is carried out in a first container, and wherein the mixture resulting from the neutralization of the inactivating agent is transferred from the first container to a second container connected to the filter system, and wherein after the mixture is transferred 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,

[0081] - in step (b), circulating the mixture through a second container and filter system until concentration is complete, and

[0082] - In step (c), the valve between the first container and the second container is opened and the second liquid is continuously directed from the first container to the second container while the mixture circulates through the filter system and the second container.

[0083] In another aspect the invention relates to a process as described and claimed herein, wherein the volume of the mixture resulting from step (a) is from 1000L to 10000L, preferably from 2000L to 8000L, and most preferably from 3000L to 5000L.

[0084] As mentioned herein, it is to be 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) before said concentration". In another aspect, the present invention relates to a method as described and claimed herein, wherein in step (b), said recombinant protein and / or said quaternary structure is finally concentrated by at least 5X, preferably at least 8X, more preferably 9X to 11X, and most preferably about 10X, compared to the volume of the mixture resulting from step (a).

[0085] The concentration step (b) of the methods provided herein can be performed such that the recombinant protein or the quaternary structure is concentrated by 3X to 50X, respectively, compared to the volume of the mixture produced from step (a) or compared to the volume of the mixture produced from step (iii). More preferably, the concentration step can be performed such that the recombinant protein or the quaternary structure is concentrated by at least 5X, such as 5X to 20X, respectively, compared to the volume of the mixture produced from step (a) or compared to the volume of the mixture produced from step (iii). Still more preferably, the concentration step can be performed such that the recombinant protein or the quaternary structure is concentrated by at least 8X, such as 8X to 14X, respectively, compared to the volume of the mixture produced from step (a) or compared to the volume of the mixture produced from step (iii). Most preferably, the concentration step is performed such that the recombinant protein or the quaternary structure is concentrated by at least 10X, such as 10X to 12X, respectively, compared to the volume of the mixture produced from step (a) or compared to the volume of the mixture produced from step (iii).

[0086] Preferably, in step (c), the total volume of second liquid added to the bulk flow is at least 5X, preferably at least 7X, more preferably at least 9X, and most preferably at least 10X the volume of the solution resulting from step (b), and / or the total volume of second liquid added to the bulk flow is most preferably approximately the volume of the mixture resulting from step (a).

[0087] In another aspect, the present invention relates to a method as described and claimed herein, wherein the method further comprises the following steps:

[0088] (d) mixing the mixture retained after step (c) with a further component selected from a pharmaceutically acceptable carrier, an adjuvant, a diluent, an excipient and a combination thereof, and wherein the concentration of the recombinant protein and / or the concentration of the quaternary structure in the solution resulting from said mixing is preferably about the concentration of the recombinant protein and / or the quaternary structure in the mixture resulting from step (a) or lower (e.g. 0.2X-0.5X) than the concentration of the recombinant protein and / or the quaternary structure in the mixture resulting from step (a).

[0089] As mentioned herein, it should be understood that "the concentration of the recombinant protein and / or the quaternary structure in the mixture resulting from step (a)" is particularly equivalent to "the initial concentration of the recombinant protein and / or the quaternary structure in the mixture in step (b) before said concentration".

[0090] 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.

[0091] "Adjuvants" as used herein may 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; water-in-oil-in-water emulsions. The emulsions may be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oils, such as squalane or squalene; oils resulting from oligomerization of olefins (particularly isobutylene or decene); esters of acids or alcohols containing linear alkyl groups, more particularly vegetable oils, ethyl oleate, propylene glycol di-(caprylate / caprate), glycerol tri-(caprylate / caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, particularly isostearates. The oil is used in combination with an emulsifier to form an emulsion. Emulsifiers are preferably nonionic surfactants, in particular sorbitan, mannide (e.g. anhydrous mannitol oleate), glycols, polyglycerols, esters of propylene glycol with oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular Pluronic products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (ed. Stewart-Tull, DES). John Wiley and Sons, NY, pp. 51-94 (1995) and Todd et al., Vaccine 15: 564-570 (1997). For example, the SPT emulsions 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 can be used. Other suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), block copolymers (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin from Escherichia coli (recombinant or otherwise), cholera toxin, IMS1314, or muramyl dipeptide, among others. Among the copolymers of maleic anhydride and alkenyl derivatives, include the copolymer EMA (Monsanto), which is a copolymer of maleic anhydride and ethylene.Dissolving these polymers in water produces an acidic solution which is preferably neutralized to physiological pH to yield an adjuvant solution into which the immunogenic, immune or vaccine composition itself is incorporated.

[0092] Another example of adjuvant is a compound selected from the polymer of acrylic acid or methacrylic acid and the copolymer of maleic anhydride and alkenyl derivative.Preferred adjuvant compounds are polymers of acrylic acid or methacrylic acid, which are especially cross-linked with polyalkenyl ethers of sugars or polyols.These compounds are known by the term carbomer (Pharmeuropa, Vol. 8, No. 2, June 1996).Those skilled in the art may also refer to U.S. Patent No. 2,909,462, which describes these acrylic polymers cross-linked with polyhydroxylated compounds, wherein the polyhydroxylated compounds have at least 3 (preferably no more than 8) hydroxyls, and the hydrogen atoms of at least 3 hydroxyls are replaced by unsaturated aliphatic groups with at least 2 carbon atoms.Preferred groups are those containing 2 to 4 carbon atoms, such as vinyl, allyl and other olefinic unsaturated groups.The unsaturated group itself may contain other substituents such as methyl.The products sold under the name Carbopol (BF Goodrich, Ohio, USA) are particularly suitable.They are cross-linked with allyl sucrose or with allyl isopentaerythritol. Among them there may be mentioned Carbopol 974P, 934P and 971 P. Most preferably Cabopol 971 P is used.

[0093] "Diluents" may include water, saline, dextrose, ethanol, glycerol, and the like.

[0094] "Isotonic agents" may include sodium chloride, dextrose, mannitol, sorbitol and lactose, among others.

[0095] "Stabilizers" include albumin and alkali metal salts of ethylenediaminetetraacetic acid, among others.

[0096] As used herein, "preservative" refers to an antimicrobial active agent, such as gentamycin, thimerosal, etc. In particular, the addition of a preservative is most preferred for the preparation of a multi-dose composition. Those antimicrobial active agents are added at a concentration that effectively prevents any microbial contamination of the composition of interest or inhibits any microbial growth within the composition of interest.

[0097] Preferably, in step (d), the further component is a combination of adjuvant, water and sodium chloride, in particular a combination of adjuvant solution and P-saline, wherein the adjuvant is preferably a polymer of acrylic acid or methacrylic acid, and still more preferably carbomer.

[0098] The term "P-saline" referred to herein particularly relates to a buffer solution consisting of 0.8-0.9% (w / v) NaCl (eg 0.85% (w / v) NaCl) dissolved in water and adjusted to pH 6.8-7.0.

[0099] In another aspect, the present invention relates to a process as described and claimed herein, wherein the mixture used in step (a) is obtainable by a procedure comprising the following steps:

[0100] (1) allowing susceptible cells in culture to be infected with a vector comprising a nucleic acid sequence encoding the recombinant protein, wherein the recombinant protein is expressed by the vector,

[0101] (2) subsequently recovering the recombinant protein and / or the quaternary structure comprising a plurality of said recombinant proteins from the cell culture medium, wherein the cell debris are preferably separated from the recombinant protein and / or the quaternary structure via a separation step (preferably comprising microfiltration through at least one filter, preferably two filters), wherein at least one filter preferably has a pore size larger than the recombinant protein and / or the quaternary structure comprising a plurality of 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.

[0102] Preferably, in the method, the separation step comprises or consists of:

[0103] - microfiltration through one or more filters with a pore size of about 2 μm to about 15 μm, and / or

[0104] - Microfiltration through one or more filters with a pore size of about 0.8 μm to about 1.0 μm.

[0105] Preferred cells are those susceptible to infection with appropriate recombinant viral vectors containing recombinant protein DNA and expressing recombinant protein. Preferably, the cells are insect cells, and more preferably, they include insect cells sold under the trademark SF+ insect cells (ProteinSciences Corporation, Meriden, CT). Preferred cell cultures have a cell count of between about 0.3-2.0×10 6 cells / mL, more preferably about 0.35-1.9×10 6 cells / mL, still more preferably about 0.4-1.8×10 6 cells / mL, even more preferably about 0.45-1.7×10 6 cells / mL, and most preferably about 0.5-1.5×10 6 cells / mL.

[0106] When used to infect susceptible cells, the multiplicity of infection (MOI) of the vector containing the nucleic acid sequence encoding the recombinant protein is preferably between about 0.03-1.5, more preferably about 0.05-1.3, still more preferably about 0.09-1.1, and most preferably about 0.1-1.0. Preferably, the MOI mentioned above refers to 1 mL of cell culture medium. Preferably, the method described herein comprises infecting 0.35-1.9×10 6 cells / mL, still more preferably about 0.4-1.8×106 cells / mL, and even more preferably about 0.45-1.7×10 6 cells / mL, and most preferably about 0.5-1.5×10 6 cells / mL, the MOI (multiplicity of infection) of the recombinant viral vector is between about 0.03-1.5, more preferably about 0.05-1.3, still more preferably about 0.09-1.1, and most preferably about 0.1-1.0.

[0107] Appropriate growth medium can also be determined by those skilled in the art, wherein a preferred growth medium is a serum-free insect cell culture medium, such as Excell 420 (JRH Biosciences, Inc., Lenexa, KS) and the like.

[0108] In the method, preferably, preferably when the recombinant protein is expressed by the vector, the cell culture in step (1) is maintained at 22-32°C, more preferably about 24-30°C, still more preferably about 25-29°C, even more preferably about 26-28°C, and most preferably 27°C, and / or the recovery in step (2) occurs 5 to 8 days, most preferably 8 days, after inoculating the cells with the vector, and / or occurs when the cell viability is reduced to less than 10%.

[0109] In another aspect, the invention relates to a method as described and claimed herein, wherein the recombinant protein is selected from:

[0110] - a recombinant PCV2 ORF2 protein, which preferably comprises, or preferably 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 to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0111] As used herein, the term "having 100% sequence identity" should be understood to be equivalent to the term "are identical".

[0112] The percentage of sequence identity has a meaning recognized in the art, and there are many methods to measure 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, ed., Computer Analysis Of Sequence Data, Part I, Humana Press, New Jersey, (1994); von Heinje, Sequence Analysis In Molecular Biology, Academic Press, (1987); and Gribskov and Devereux, ed., Sequence Analysis Primer, M Stockton Press, New York, (1991). Methods for aligning polynucleotides or polypeptides are encoded in computer programs, including the GCG 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 using the FASTA algorithm can be used, wherein the gap opening penalty for affine gap searches is -12 and the gap extension penalty is -2. For the purposes of the present invention, protein / amino acid sequences were aligned using the Clustal W method with DNASTAR Inc.'s MegAlign software version 11.1.0 (59), 419 using the default multiple alignment parameters set in the program (Gonnet series protein weight matrix with gap penalty = 10.0, gap length penalty = 0.2, and delayed divergent sequence (%) = 30%).

[0113] As used herein, it is particularly to be understood that the term "sequence identity to the sequence of SEQ ID NO: X" is equivalent to the term "sequence identity to the sequence of SEQ ID NO: X over the length of SEQ ID NO: X" or the term "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 one of the SEQ ID NOs mentioned herein.

[0114] It is further understood that the term "recombinant protein consisting of a sequence" particularly also relates to any one or more co-translational and / or post-translational modifications of the sequence affected by the cell (wherein the polypeptide is expressed). Thus, as described herein, the term "recombinant protein consisting of a sequence" also relates to a sequence having one or more modified sequences affected by the cell (wherein the polypeptide is expressed), particularly modifications of amino acid residues affected in protein biosynthesis and / or protein processing (preferably selected from glycosylation, phosphorylation and acetylation).

[0115] In another aspect, the present invention relates to a method as described and claimed 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.

[0116] In another aspect, the present invention relates to a method as described and claimed herein, wherein the virucidal activity of the mixture produced from said method is reduced by at least 20% compared to a mixture which has not been subjected to the concentration of step (b) and the continuous diafiltration of step (c) of said method, and / or wherein when live virus is mixed with the immunogenic composition produced by said method for 4 hours or more, in particular for 4 hours or more at room temperature, said immunogenic composition results in less than 1 logTCID 50 / mL loss of live virus. "Room temperature" as referred to herein particularly relates to 20 to 25°C, and more particularly (on average) 23°C.

[0117] In another aspect, the invention relates to a method as described and claimed herein, wherein the live virus is Porcine Reproductive and Respiratory Syndrome (PRRS) virus.

[0118] In another aspect, the present invention relates to a method as described and claimed herein, wherein the method further comprises the step of combining the mixture retained 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 particularly to be understood that the term "retained after a step" is equivalent to "resulting from a step".

[0119] The present application not only provides methods of preparing immunogenic compositions, it also relates to immunogenic compositions prepared by the methods as described and claimed herein.

[0120] In another aspect, the present invention therefore relates to an immunogenic composition obtainable by a method as described and claimed herein.

[0121] According to a particularly preferred aspect, the immunogenic composition is a solution comprising 1-10 μM L-arginine and / or 1-10 μM L-lysine, and most preferably, the immunogenic composition is a solution comprising 1-10 μM L-arginine and 1-10 μM L-lysine.

[0122] Therefore, the present invention also provides an immunogenic composition comprising:

[0123] - a recombinant PCV2 ORF2 protein, preferably consisting of or comprising 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, and

[0124] - 1-10 μM L-arginine and / or 1-10 μM L-lysine,

[0125] And wherein optionally, the immunogenic composition is substantially free of neutralized inactivating agent and / or substantially free of neutralizing agent.

[0126] It is to be understood that "1-10 μM" as used herein is specifically equivalent to "from 1 μM up to 10 μM".

[0127] In particular, the immunogenic composition or mixture resulting from step (d) comprises 4-400 μg of recombinant PCV2 ORF2 per ml, or preferably 8-200 μg of recombinant PCV2 ORF2 protein per ml.

[0128] "Substantially free of neutralizing agents" is particularly to be understood as meaning that the immunogenic composition comprises less than 150 nM of neutralizing agents. Thus, for example, the indication that "the immunogenic composition is substantially free of sodium thiosulfate" is particularly to be understood as being equivalent to the statement that "the immunogenic composition comprises less than 150 nM of sodium thiosulfate".

[0129] According to a further aspect, the present invention therefore relates to an immunogenic composition obtainable by the process as described and claimed herein, wherein the immunogenic composition or mixture resulting from step (d) is essentially free of sodium thiosulfate or comprises less than 150 nM sodium thiosulfate.

[0130] Furthermore, there is provided an immunogenic composition, in particular an immunogenic composition obtainable by a process as described and claimed herein, preferably an immunogenic composition obtainable by any of said processes comprising step (d), wherein said immunogenic composition comprises

[0131] 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 to the sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2, and

[0132] 0.1-1 μM L-tryptophan; and / or

[0133] 0.1-3 μM L-glutamine; and / or

[0134] 0.2-4 μM L-methionine; and / or

[0135] 1-10 μM L-arginine; and / or

[0136] 1-10 μM L-threonine; and / or

[0137] 1-15 μM L-lysine.

[0138] Preferably, the immunogenic composition as described and claimed herein comprises

[0139] 0.1-0.7 μM L-tryptophan; and / or

[0140] 0.7-2.4 μM L-glutamine; and / or

[0141] 0.5-3.2 μM L-methionine; and / or

[0142] 1.4-4.7 μM L-arginine; and / or

[0143] 1.6-8.0 μM L-threonine; and / or

[0144] 4.0-12.2 μM L-lysine.

[0145] More preferably, the immunogenic composition as described and claimed herein comprises

[0146] 0.2-0.6 μM L-tryptophan; and / or

[0147] 0.8-2.2 μM L-glutamine; and / or

[0148] 0.8-2.2 μM L-methionine; and / or

[0149] 1.6-4.3 μM L-arginine; and / or

[0150] 2.4-6.3 μM L-threonine; and / or

[0151] 4.4-11.2 μM L-lysine.

[0152] According to particularly preferred aspects, the immunogenic composition as described and claimed herein comprises 0.7-2.4 μM L-glutamine and 4.0-12.2 μM L-lysine, or preferably comprises 0.8-2.2 μM L-glutamine and 4.4-11.2 μM L-lysine.

[0153] Preferably, the immunogenic compositions as described and claimed herein are substantially free of sodium thiosulfate.

[0154] According to a further preferred aspect, there is provided an immunogenic composition, in particular obtainable by a method as described and claimed herein, preferably obtainable by any of said methods comprising step (d), wherein when subjected to two-dimensional (2D) ultra performance liquid chromatography (UPLC) at room temperature exhibits a ratio of Peak A area to Peak B area of ​​0.2-0.4 and / or a ratio of Peak C area to Peak B area of ​​0.5-0.9, wherein the 2D UPLC comprises

[0155] The first dimension of chromatography has a system volume of 400 μL, where

[0156] - injecting a sample comprising the immunogenic composition into an anion exchange column wherein 0.1 mL of the column volume is filled with a quaternary amine functionalized material suitable for binding virus-like particles, and

[0157] - a multi-step gradient method driving an anion exchange column with an initial 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 to 10% 50 mM Tris (pH 8) and 90% 50 mM Tris, 2 M NaCl (pH 8) at 2 min at a flow rate of 0.6 mL / min,

[0158] And at a retention time of 2.67 min, it switches from the first dimension of the chromatography to

[0159] The second dimension of chromatography, in which 50 μL of the eluate from the first dimension of chromatography was driven at a flow rate of 0.3 mL / min through a column having a 1.75 mL column volume and A size exclusion column with a pore size of

[0160] and wherein the chromatogram is recorded by monitoring the fluorescence emission at a wavelength of 330 nm and an excitation wavelength of 280 nm, and wherein

[0161] Peak A area is the peak area of ​​the highest peak in the chromatogram with a retention time of 0-1 min;

[0162] Peak B area is the peak area of ​​the highest peak with a retention time of 7-8 minutes in the chromatogram;

[0163] The peak C area is the peak area of ​​the highest peak at a retention time of 8.5 to 9.5 minutes in the chromatogram.

[0164] According to a preferred aspect, the sample comprises the immunogenic composition diluted in 50 mM Tris, pH 8. For example, if the sample comprises the immunogenic composition retained after step (c), the sample preferably comprises or consists of the immunogenic composition diluted 1:10 in 50 mM Tris, pH 8.

[0165] Quaternary amine functionalized materials suitable for binding virus-like particles are preferably macroporous and / or non-porous materials.

[0166] Example 5 describes this 2D UPLC method which can be used by a person skilled in the art. In case of controversial results and any doubts, the ratios of peak areas as mentioned herein refer to those estimated by / can be estimated by the method as described in Example 5.

[0167] The term "immunogenic composition" means, but is not limited to, a composition of a substance comprising at least one antigen, which elicits a cellular and / or antibody-mediated immune response in a host to an antigen of interest. Typically, an "immune response" includes, but is not limited to, one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, inhibitory T cells, and / or cytotoxic T cells and / or γ-δ T cells specifically directed to one or more antigens included in the composition or vaccine of interest. Preferably, the host will present a therapeutic or protective immune response, so that resistance to new infections will be enhanced and / or the clinical severity of the disease will be reduced. In such cases, the immunogenic composition is a "vaccine". The protection will be exhibited by reducing or lacking symptoms commonly presented by infected hosts, faster recovery times, and / or reduced viral titers in infected hosts. The host as described herein is particularly a mammal, preferably a pig.

[0168] In another aspect, the virucidal activity of the immunogenic composition prepared by the method as described and claimed herein is reduced by at least 10%, preferably at least 20%, compared to a corresponding immunogenic composition that has not undergone concentration of step (b) and 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 a corresponding immunogenic composition that has not undergone concentration of step (b) and continuous diafiltration of step (c). Still more preferably, the virucidal activity of the immunogenic composition is reduced by at least 70% compared to a corresponding immunogenic composition that has not undergone concentration of step (b) and continuous diafiltration of step (c). Even more preferably, the virucidal activity of the immunogenic composition is reduced by at least 90% compared to a corresponding immunogenic composition that has not undergone concentration of step (b) and continuous diafiltration of step (c).

[0169] For the purposes of the present invention, the term "virucidal activity" means that when a liquid, fluid, solution, composition, etc. is mixed with live viruses or live bacteria, the liquid, fluid, solution, composition, etc. inactivates or kills these live viruses or live bacteria to a certain extent. Therefore, a reduction of at least 10% in the virucidal activity of a liquid, fluid, solution, composition, etc. means that the survival rate of live viruses or live bacteria in a liquid, fluid, solution, composition, etc. that has undergone any of these preparation methods is 90% higher than that of a liquid, fluid, solution, composition, etc. that has not undergone any of the production methods described herein. In this context, "survival rate" particularly relates to the percentage of viruses or bacteria that maintain replication ability in a host.

[0170] According to a particularly preferred aspect, when live virus or live bacteria is mixed with an immunogenic composition prepared by the method as described and claimed herein and incubated for at least 4 hours, the immunogenic composition results in less than 1 log TCID 50More preferably, when live virus or live bacteria are mixed with the immunogenic composition prepared by the method as described and claimed herein and incubated for at least 4 hours, the immunogenic composition results in less than 0.9 log TCID 50 Even more preferably, when live virus or live bacteria are mixed with the immunogenic composition prepared by the method as described and claimed herein and incubated for at least 4 hours, the immunogenic composition results in less than 0.7 log TCID 50 Still more preferably, when live virus or live bacteria are mixed with the immunogenic composition prepared by the method as described and claimed herein and incubated for at least 4 hours, the immunogenic composition results in less than 0.5 log TCID 50 Even more preferably, when live virus or live bacteria are mixed with the immunogenic composition prepared by the method as described and claimed herein and incubated for at least 4 hours, the immunogenic composition results in less than 0.3 log TCID 50 / mL of live virus or less than 0.3 log CFU / mL of live bacteria.

[0171] The TCID per milliliter can be estimated by a standard in vitro titration assay that allows estimation of the amount of viable virus. 50 The CFU per milliliter can also be determined by a standard in vitro titration assay which allows the amount of viable bacteria to be estimated. The term "per milliliter" preferably refers to at least 1 mL of fluid.

[0172] Furthermore, it has been found that the immunogenic compositions obtainable by the process as described and claimed herein are particularly stable.

[0173] According to a particular preferred aspect, the shelf life of the immunogenic composition prepared by the method as described and claimed herein is at least 2 years, preferably at least 3 years, wherein the term "shelf life" as used herein means the period of time over which the product can be stored without the quality decreasing below a certain minimum acceptable level.

[0174] In particular, the present invention relates to an immunogenic composition, wherein upon administration of one dose of the immunogenic composition, the immunogenic composition preferably induces a protective immune response in pigs against a pathogen (from which the amino acid sequence of the recombinant protein is derived), wherein the pathogen is preferably PCV2, and wherein the administration preferably administers one dose of the immunogenic composition to pigs.

[0175] In another aspect, the present invention relates to an immunogenic composition as described and claimed herein, wherein the immunogenic composition further comprises an attenuated live virus, preferably an attenuated porcine reproductive and respiratory syndrome (PRRS) virus or an attenuated live bacteria.

[0176] For the purposes of the present invention, a "live" virus or bacterium refers to a virus or bacterium that is capable of replicating in a host. Preferred live viruses and preferred live bacteria of the present invention are PRRS virus (PRRSV) and Mycoplasma hyopneumoniae bacteria, respectively. However, the terms live viruses or live bacteria, respectively, are not limited to PRRSV and Mycoplasma hyopneumoniae.

[0177] In another aspect, the present invention relates to an immunogenic composition as described and claimed herein, wherein the immunogenic composition further comprises an attenuated porcine reproductive and respiratory syndrome (PRRS) virus.

[0178] The term "attenuation" particularly relates to viruses or bacteria that have been cultured in a manner that reduces their virulence and makes them suitable for use as vaccines. Thus, more specifically, "attenuation" relates to live viruses or bacteria, but which are no longer able to produce disease due to reduced virulence or toxicity through these special cultures.

[0179] Preferably, upon administration of one dose of the immunogenic composition, said immunogenic composition induces a protective immune response against PRRS virus, particularly in pigs, and wherein said administration preferably administers one dose of the immunogenic composition to pigs.

[0180] More preferably, upon administration of one dose of the immunogenic composition, said immunogenic composition induces a protective immune response in particular against PCV2 and PRRS virus in pigs, and wherein said administration preferably administers one dose of the immunogenic composition to pigs.

[0181] In another aspect, the present invention also provides a kit, wherein the kit comprises a container containing the immunogenic composition as described and claimed herein, and wherein the kit preferably further comprises at least one additional container containing at least one additional antigen selected from a live attenuated virus (preferably an attenuated PRRS virus) and a live attenuated bacterium.

[0182] According to yet another aspect, there is provided a kit comprising a container containing an immunogenic composition as described and claimed herein.

[0183] The immunogenic composition as described and claimed herein and / or the kit as described and claimed herein may further comprise at least one additional antigen, preferably a viral or bacterial antigen, and even more preferably a viral or bacterial antigen from at least one other pathogen causing disease in pigs. The additional antigen may be any of those disclosed in International Patent Application WO2007 / 094893, the contents and teachings of which are incorporated herein by reference. In short, the additional antigen may be an antigen of any other pathogen causing disease in pigs. Preferably, "any other pathogen causing disease in pigs" or at least one other pathogen causing disease in pigs is selected from: Actinobacillus pleuropneumonia (1); adenovirus (2); alphaviruses, such as Eastern equine encephalomyelitis viruses (3); Bordetella bronchiseptica (4); Brachyspira spp. (5), preferably B. hyodysentheriae (6); B. piosicoli (7), Brucella suis, preferably biovar 1, 2 and 3 (8); Classical swine fever virus (9); Clostridium spp. (10); spp.) (10), preferably Clostridium difficile (Cl.difficile) (11), Clostridium perfringens (Cl.perfringens) (12), Clostridium novyi (Cl.novyi) (13), Clostridium septicum (Cl.septicum) (14), Clostridium tetani (Cl.tetani)(15); Coronavirus(16), preferably Porcine Respiratory Coronavirus(17); Eperythrozoonosis 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 spp.(25), preferably Leptospira australis(26); Leptospira canis(27); canicola)(27); Leptospira grippotyphosa)(28); Leptospira icterohaemorrhagicae(29); and Leptospira interrogans(30); Leptospira pomona(31); Leptospira tarassovi(32); Mycobacterium spp.(33) preferably Mycobacterium avium(34), Mycobacterium intracellulare(35) and Mycobacterium bovis(36); Mycoplasma hyopneumoniae(37); Pasteurella multocida(38); Porcine cytomegalovirus(39); cytomegalovirus (39); Porcine Parvovirus (40); Porcine reproductive and respiratory syndrome virus (41); Pseudorabies virus (42); Rotavirus (43); Salmonella spp. (44), preferably S. thyhimurium (45) and Salmonella choleraesuis (S.choleraesuis)(46); Staph.hyicus(47); Staphylococcus spp.(48), preferably Streptococcus spp.(49), preferably Streptococcus suis(50); Swine herpes virus(51); Swine Influenza Virus(52); Swinepox virus(53); Swine pox virus(54); Vesicularstomatitis virus(55); Swine vesicular exanthema virus(56); Leptospira Hardjo(57); Mycoplasma hyosynoviae(58); and / or combinations thereof.

[0184] Most preferably, the immunogenic composition as described and claimed herein and / or the kit as described and claimed herein further comprises one or more attenuated or inactivated non-PRRSV pathogens or antigenic material thereof.

[0185] In another aspect, the present invention relates to an immunogenic composition as described and claimed herein and / or a kit as described and claimed herein for use as a medicament, preferably as a vaccine.

[0186] In another aspect, the present invention relates to an immunogenic composition as described and claimed herein and / or a kit as described and claimed herein for use in an animal, preferably a pig, in the following method:

[0187] - Induce a protective immune response against at least one pathogen, and / or

[0188] -Reduction of one or more clinical signs of infection with at least one pathogen.

[0189] The present invention further provides the following methods in animals:

[0190] - Induce a protective immune response against at least one pathogen, and / or

[0191] - Reduction of one or more clinical signs of infection with at least one pathogen

[0192] Therein the immunogenic composition as described and claimed herein is administered to an animal, in particular to a pig.

[0193] According to a further aspect, the present invention relates to the use of an immunogenic composition as described and claimed herein for the preparation of a medicament for use in animals, preferably in pigs.

[0194] - Induce a protective immune response against at least one pathogen, and / or

[0195] - Reduction of one or more clinical signs of infection with at least one pathogen.

[0196] Preferably, the at least one pathogen is selected from the group consisting of: PCV2, PRRSV, non-PRRSV pathogens, and combinations thereof.

[0197] The term "non-PRRSV pathogen" as used herein particularly relates to a pathogen selected from the group consisting of Mycoplasma hyopneumoniae, pseudorabies virus, swine influenza virus, porcine parvovirus, Transmissibie gastroenteritis virus, Escherichia coli, Erysipelothrix, Bordetella bronchiseptica, Salmonella choleraesuis, Haemophilus parasuis, Pasteurella multocida, Streptococcus suis and Actinobacillus pleuropneumoniae.

[0198] As used herein, the term "reducing one or more clinical signs" shall particularly mean that when both the animal and the "control group" animal are infected or challenged with a pathogen (the (one or more) immunogenic active ingredients in the immunogenic composition are derived from the pathogen), the incidence or severity of any of these signs in the animal receiving the immunogenic composition is reduced compared to the "control group" animal, and wherein the control group does not receive the administration of the immunogenic composition. In this context, the term "reducing" 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.

[0199] As used herein, "clinical signs" shall refer to signs from pathogen infection that can be directly observed from a living animal, such as symptoms. Representative examples will depend on the pathogen selected, but may include, for example, runny nose, lethargy, coughing, high fever, reduced weight gain or weight loss, dehydration, diarrhea, swelling, lameness, etc.

[0200] As used herein, a "protective immune response" refers to a reduction in the incidence or severity of clinical, pathological, or histopathological signs or symptoms from infection with the pathogen of interest, up to and including complete prevention of such signs or symptoms. Example

[0201] Embodiment 1:

[0202] Preparation of PCV2 ORF2 protein - Upstream processing

[0203] PCV2 ORF2 protein was prepared in baculovirus infected SF+ cells in a method corresponding to that described in WO 2006 / 072065 A2, Examples 1 to 3. In the following, this method is outlined:

[0204] PCV2 ORF2 baculovirus-vectors were prepared in a bioreactor. The culture medium was pre-sterilized or sterile filtered and added to the bioreactor. SF+ cells from the expansion culture were added to the culture medium. When inoculating PCV2 ORF2 seeds, the cells were inoculated at the same time (simultaneous infection). The temperature was maintained at 27±2°C and the pH was monitored throughout the virus propagation process. Dissolved oxygen (DO) was controlled by sparging clean compressed air and oxygen (O2). The harvest window occurred between 6 and 8 days after virus infection and the harvest criterion of ≤20% cell viability was reached. At harvest, two sets of filters (i.e., a pre-filter with a pore size of 2.0-15.0 μm and a final filter with a pore size of 0.8-1.0 μm) were used to clarify the PCV2 ORF2 antigen fluid. The filtered harvest fluid was collected in a tank. For the inactivation of baculovirus, 5 mM BEI was added for up to 96 hours before neutralization. For the neutralization of the remaining BEI, 5 mM sodium thiosulfate was added to neutralize the BEI. The addition of sodium thiosulfate occurred 72-96 hours after inactivation.

[0205] Preparation of PCV2 ORF2 protein - Downstream processing

[0206] Laboratory scale

[0207] The 3L sample generated by the above upstream process was sent to the laboratory and concentrated to 300mL based on volume to achieve a 10-fold concentration. After concentration, the antigen was then diafiltered using 3L of P-saline, a buffer solution consisting of 0.85% NaCl dissolved in water and adjusted to pH 6.8-7.0.

[0208] Laboratory-scale concentration and continuous diafiltration were performed under a biosafety cabinet or BSC at room temperature (27 ± 2 °C) using a tangential flow filter (TFF) system consisting of a TFF System Sartoflow Slice 200 benchtop system equipped with one Sartoslice Ultrafilter (UF) cassette filter (PES, 200 cm 2 Filtration area, 300 kD cut-off).

[0209] Before ultrafiltration / diafiltration, rinse the TFF system (at least 1 hour) with 1M NaOH, and sterilize the box chemically overnight with 0.1NaOH. Then, put the sterilized system and filter into BSC for aseptic assembly, and connect inlet, outlet and permeate pipeline. The permeate pipeline is connected to the waste bottle, and the inlet pipeline is connected to the inactivated antigen to be concentrated simultaneously. The outlet pipeline is returned to the retention bottle from the filtration unit. Use sterile PBS (each filter 10L) balance filter.

[0210] The product is concentrated to 10 times of the original antigen volume based on volume by ultrafiltration. When the antigen is processed by concentration, the flow rate through the filter changes. At a small scale, the flow rate is a range and is maintained by placing a back pressure on the system. Diafiltration is performed with P-saline with 10 times the volume of the ultrafiltration volume, wherein the P-saline is added at a rate equal to the permeate flow. The final concentrated and diafiltered antigen is stored at 2-8 ℃.

[0211] Using HPLC quantification methods, it was seen that this process had eliminated ≥99% of any residual insect cell culture medium (ExCell 420 medium) and reduced neutralized BEI and residual sodium thiosulfate to undetectable levels.

[0212] Furthermore, preliminary experiments have shown that in the context of preparing immunogenic compositions, as described herein, the use of flat plate filters for TFF systems has the beneficial effect of significantly reducing storage costs as compared to hollow fiber filters, since fewer storage bags / drums (200 L) need to be utilized. Additionally, it can be seen that by using flat plate filters, labor and preparation time for harvesting are significantly reduced.

[0213] Operation scale

[0214] In operation or large-scale processing (see Figure 1 ), using about 4000 L produced by the upstream process described above. The system is designed so that the solution containing the antigen 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.

[0215] The DSP tank is a repository for antigen and final antigen presentation in the method. The programmed weight of this tank controls the entire system, because when certain programmed weights are reached, the method will proceed forward. In addition, this tank is where the final antigen product is collected and harvested in a 200L bucket for storage.

[0216] The DSP tank is connected to a microfiltration (MF) unit. After the solution containing the antigen is sent 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 transferred to waste.

[0217] The method begins with obtaining the weight of the inactivation tank containing the (intermediate) product generated by the upstream process. A 10-fold concentration is calculated based on the initial weight of the tank and programmed into the system operating unit of the MF unit. Once concentration begins, the system triggers the transfer line between the inactivation tank and the DSP tank to open or close based on the tank weight. When the inactivation tank becomes empty, the system triggers the tank to close the transfer line to the DSP tank. After the inactivation tank is empty, a separate transfer line is started to add brine to the recently evacuated inactivation tank. The brine is also monitored by weight because the desired amount of brine transferred to the DSP tank is the original weight obtained and used for concentration calculation. The retentate is fed back to the DSP tank and further passed through the MF unit again to further concentrate the retentate until the weight of the retentate is reduced to a programmed value indicating the desired concentration.

[0218] The MF unit is unique in that it can support both microfiltration and ultrafiltration cartridges. For this procedure, a total of 6 ultrafiltration sartocubes (21m3 each) 2 ) is placed in the MF unit. This surface area was selected after extensive evaluation of the system. There is a feed line from the DSP tank into the MF unit, a retentate line that feeds back to the DSP tank, and a transfer line from the permeate to the waste liquid. This unit is programmed to run at a permeate pressure of 0psi while maintaining a recirculation of 20-35psi, with a target of a transmembrane pressure (TMP) of 29psi. The recirculation pump runs at 100% capacity until the holding mode is activated. During the concentration and diafiltration process, these parameters are targeted. Once the DSP tank is at a set weight, the MF unit is activated to start the concentration step, and the antigen will be kept circulating until the set 10-fold concentration is reached. Once the 10-fold concentration is completed, the MF unit is switched to the holding mode so that the antigen is circulated at a reduced pressure and a reduced speed while the inactivation tank is filled with P-brine. After the P-brine reaches the desired volume as the original weight at the start in the inactivation tank, the system will start the diafiltration process by starting the P-brine flow into the DSP tank, and the MF unit will ramp up back to the target range.

[0219] 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 10 times penetration is achieved. The concentrated and diafiltered antigen is in the DSP tank and pumped into the antigen harvest bucket for final storage. These buckets are stored at 4±2°C until vaccine blending.

[0220] The products resulting from the above upstream and downstream processes are then finally mixed with a carbomer solution (adjuvant) and normal saline (P-saline). This investigational veterinary product is also referred to hereinafter as Investigational Veterinary Product No. 1 (IVP) or "IVP1", respectively.

[0221] In summary, specific advantages observed with respect to the novel process described herein are time savings, reduced handling, and storage / space savings in the cooler.

[0222] Embodiment 2:

[0223] In-use stability studies

[0224] Overview

[0225] Using the optimized protocol, a study was designed to investigate the in-use stability of an investigational veterinary product (lyophilisate) (comprising modified live PRRS virus) following reconstitution with Investigational Veterinary Product No. 1 (IVP1) of Example 1 (the investigational vaccine product also referred to hereinafter as "IVP2").

[0226] A summary of the investigational veterinary products (IVPs) used is provided in Table 1.

[0227] The evaluation included two batches of IVP1 (one prepared at high potency and one prepared at conventional / standard release potency) and two batches of IVP2.

[0228] All investigational veterinary products (IVPs) tested (see Table 1) were stored at 5°C ± 3°C until used in the study.

[0229] In this study, each batch of IVP2 vaccine was reconstituted with IVP1 and PRRS virus titers were tested at 0, 2 and 4 hours after reconstitution.

[0230] 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 washing solution)) or with IVP1 and regardless of the scientist. In addition, after 2 hours and 4 hours of incubation at room temperature, the test results showed no change to negligible change in the virus titer.

[0231] The results of the study support the relevant use of IVP1 and IVP2.

[0232] 1. Introduction

[0233] The study was designed to demonstrate the in-use stability of IVP2 after reconstitution with IVP 1. IVP2 was a freeze-dried modified live vaccine (MLV) and IVP 1 was prepared as described above.

[0234] In the following, the results of the in-use stability assessment of IVP2 after reconstitution with IVP1 are shown.

[0235] 2. Goals / Objectives of the Study

[0236] The purpose of this in-use stability study was to investigate the stability of the IVP2 vaccine after reconstitution with IVP1.

[0237] 3. Materials

[0238] Two lots of each of the two vaccines evaluated in the study were used, along with one lot of diluent control, with the composition of the test materials listed in Table 1.

[0239] Table 1. Test material composition

[0240]

[0241] 4. Study Design

[0242] Each batch of IVP2 vaccine was tested in all possible combinations with each batch of subunit vaccine (IVP1). Table 2.1 lists all 8 vaccine combinations evaluated in this study.

[0243] Table 2.1 List of combinations of IVPs evaluated

[0244]

[0245] The PRRS virus titer of the samples was tested immediately after reconstitution and merging at time zero (T0). The merged samples were stored in the dark at room temperature and tested again at 2 hours (T2) and 4 hours (T4). The samples were prepared on the same day of the test. Each sample was tested at two different time periods, and the time periods were performed on different days.

[0246] Testing was performed by two scientists. Scientist 1 tested samples #1 to #4 with IVP2 batch #1, and Scientist 2 tested samples #5 to #8 with IVP2 batch #2. Each scientist tested two samples per time period and one in-use stability control. The control consisted of IVP2 reconstituted with sterile washed phosphate buffered saline (WPBS) using the same volumes and time intervals as indicated for the corresponding test samples.

[0247] 5. Methods

[0248] 5.1 Reconstruction Method

[0249] For each sample (as described in Table 2.1), each of the three IVP2 vials was reconstituted with 10 mL of IVP1. For each control, each of the three IVP2 vials was reconstituted with 10 mL of WPBS. After reconstitution, the contents of each of the three vials were combined before starting the test, as described above.

[0250] 5.2 Test Method

[0251] Day -3

[0252] A cell culture suspension of AK-MA104 cells was prepared in MEM supplemented with 10% FBS and gentamicin sulfate. 100 μL of the cell culture was inoculated into each well of a 96-well microtiter plate. The plate was closed with a lid and kept in an incubator for 3 days.

[0253] Day 0

[0254] Sample preparation

[0255] Samples of IVP1 and the positive control were used to resuspend the lyophilized sample of IVP2. Three vials were combined and the pool was titrated in triplicate.

[0256] Titration

[0257] MEM supplemented with 2% FBS and gentamicin sulfate was used as dilution medium for diluting the samples.

[0258] Vaccination

[0259] The wells for the samples were inoculated with 100 μL of the diluted samples, and the wells for the negative controls were inoculated with 100 μL of culture medium.

[0260] Incubation

[0261] This was incubated in an incubator for 8 days.

[0262] Day +8

[0263] Read and calculate

[0264] The final cytopathic effect (CPE) readings of the plates were read under an inverted microscope and the raw data were converted to titers per 1 mL dose by adding 1.0 log.

[0265] The titer was 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 log10TCID50 / 1mL 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 periods, with three independent titrations performed for each period.

[0266] 6. Acceptance of the Guidelines

[0267] Virus titers of IVP2 obtained at 0 (T0), 2 (T2) and 4 (T4) hours after reconstitution with WPBS diluent control were greater than or equal to the predetermined / previously determined minimum immunizing dose (MID).

[0268] 7. Results

[0269] A summary of the mean titers of the test samples and each control obtained at each incubation time (T0, T2, and T4) and the corresponding titer differences relative to time 0 (T0-T0, T2-T0, and T4-T0) are shown in Tables 3 and 4, respectively.

[0270] Table 3. Average virus titers

[0271]

[0272] Table 4. Differences in mean viral titers relative to T0

[0273]

[0274] PRRS virus titer expressed as log 10 TCID 50 Each potency value represents the mean of the results obtained on two test days, with three independent titrations performed on each test day.

[0275] 8. Discussion

[0276] The data obtained in this study showed that the viral titers of IVP2 rehydrated with standard diluent (WPBS) were all above the MID and remained stable during the four-hour incubation at room temperature. These results are consistent with the known previously determined product characteristics of IVP2 and confirm the validity of this in-use stability assessment of the modified live PRRS virus vaccine IVP2 used in combination with IVP1.

[0277] Results obtained with both lots of IVP2 tested with either lot of IVP1 at T0 showed viral titers comparable to those obtained with each control. The variability observed between control and test samples was within the range of analytical variability seen in each analytical validation study.

[0278] The results obtained with the two lots of IVP2 after reconstitution with either lot of IVP1 at T2 and T4 showed stable viral titers without a minimal decrease in titer (≤0.12 log10 TCID50).

[0279] 9. Conclusion

[0280] The results of this study support the combined use of IVP1 and IVP2, and the in-use stability of IVP2 when mixed with IVP1 for 4 hours.

[0281] Embodiment 3:

[0282] A) Initiation of immunity to PRRSV challenge

[0283] The aim of this study was to investigate whether the onset of immunity of IVP2 at 3 weeks post-vaccination would be affected when used in combination with IVP1. For this purpose, the IVP2 vaccine was reconstituted to the minimum immunizing dose (MID) with IVP1 prepared with high potency, wherein this combination of IVP1 and IVP2 is also referred to as "IVP1 / IVP2" hereinafter. This study included 52 three-week-old piglets from a commercial herd in Germany. The main parameter studied was lung lesions at necropsy, 10 to 12 days post-challenge.

[0284] At inclusion, none of the animals were antibody or antigen positive to PRRSV. Until challenge with PRRSV (on day 21 after vaccination), none of the animals in the control group showed seroconversion or tested positive for PRRSV antigen.

[0285] The median lung lesion score of animals vaccinated with IVP1 / IVP2 was reduced by 52.1% compared to animals in the control group. In addition, since animals vaccinated with IVP1 / IVP2 had a statistically significant higher ADWG of 164 grams per day after challenge compared to the control group, the ADWG results support the efficacy of the combination of IVP1 and IVP2.

[0286] In animals vaccinated with IVP1 / IVP2, PRRSV antibodies were first detected 14 days after vaccination (D14). Although there was no statistically significant difference in the number of PRRSV antibody-positive animals between the two groups one week after the challenge, the antibody levels were significantly higher in the vaccinated group compared to the control group. Ten to twelve days after the challenge, all animals were PRRSV antibody-positive, and there was no statistically significant difference in the antibody content between the groups.

[0287] In conclusion, it was demonstrated that the onset of immunity of IVP2 at 3 weeks post-vaccination was not negatively affected when used in combination with IVP1.

[0288] B) Onset of immunity against PCV2 challenge

[0289] The aim of this study was to evaluate the onset of immunity of IVP1 when used in combination with IVP2. For this purpose, the IVP2 vaccine was reconstituted to target a maximum immunizing dose by adding IVP1 formulated to target a minimum efficacy, wherein this mixture of IVP1 and IVP2 is also referred to hereinafter as "IVP2 / IVP1".

[0290] The study included 60 Caesarean-derived, colostrum-deprived (CDCD) pigs that were seronegative for PCV2, of which 30 were vaccinated with IVP2 / IVP1 and 30 (control group) received sterile diluent (water for injection) at 3 weeks of age (i.e., on study day 0 (D0)) followed by a virulence challenge against PCV2 on D15.

[0291] Vaccination with IVP2 / IVP1 resulted in a significant increase in pigs that were serologically positive for PCV2. Within one week after challenge, 28 pigs (93%) from the IVP2 / IVP1 group seroconverted to positive levels, while 0 / 30 pigs (0%) from the control group were positive. Animals from the control group began to seroconvert to positive levels 13 days after challenge. On D21, D 28 and D35, the IVP2 / IVP1 group had a significantly higher proportion of pigs with positive PCV2 titers compared to the control group. As of D42, the IVP2 / IVP1 group was 100% serologically positive for PVC2, while the control group was 92% positive.

[0292] When evaluating the primary outcome parameters, vaccination with IVP2 / IVP1 significantly reduced lymphoid depletion, lymphoid inflammation, and PCV2 lymphoid colonization compared to the control group. In addition, the overall level of histological lesions was more severe in the control group, with 23 / 30 pigs (76.7%) having moderate to severe scores in at least one category of lymphoid lesion assessment, while only 2 / 30 pigs (6.7%) vaccinated pigs had moderate lymphoid lesion scores, none of which were severe.

[0293] In conclusion, IVP1 formulated with minimal efficacy and used in combination with IVP2 provided effective active immunity to 3-week-old CDCD pigs when challenged with virulent PCV2 on day 15 post-vaccination.

[0294] Furthermore, in further vaccination-PCV2 challenge studies, in which monovalent IVP1 was compared with respective products that had not been subjected to downstream processing according to Example 1 (the products are referred to herein as "IVPO"), IVP1 was observed to be at least as effective as the established IVP0.

[0295] C) Duration of immunity against PCV2 challenge

[0296] The purpose of this study was to evaluate the duration of immunity of IVP1 when used in combination with IVP2. For this purpose, the IVP2 vaccine was reconstituted to target the maximum immunizing dose by adding IVP1 formulated to target minimum efficacy. As mentioned above (under B), this mixture of IVP1 and IVP2 is also referred to herein as "IVP2 / IVP1".

[0297] The study included 60 Caesarean-derived, colostrum-deprived (CDCD) pigs that were seronegative for PCV2, of which 30 were vaccinated with IVP2 / IVP1 and 30 (control group) received sterile diluent (water for injection) at 3 weeks of age (i.e., on study day 0 (D0)) followed by a virulent challenge with PCV2 on D119 (17 weeks duration of immunity).

[0298] Thus, animals vaccinated with IVP2 / IVP1 had significantly reduced frequencies of lymphoid depletion, lymphoid colonization, and viremia after PCV2 challenge compared to control animals. In addition, animals vaccinated with IVP2 / IVP1 had significantly reduced PCV2 viral loads on all sampling days at a median of 28 days after PCV2 challenge, with no viral levels considered viremic enough to cause disease ((rt-PCR) test ≥1.0×10 4 PCV2 genome equivalents), while control animals were viremic. Vaccination with IVP2 / IVP1 resulted in a significant increase in pigs seropositive for PCV2 from vaccination (D0) to D133 (two weeks after challenge with PCV2), which has historically been a strong indicator of efficacy. The majority of the control group (81%) was not seropositive for PCV2 until D140 (3 weeks after challenge with PCV2).

[0299] These results show that the duration of immunity of IVP1 at 17 weeks post-vaccination was not negatively affected when used in combination with IVP2.

[0300] D) Duration of immunity against PRRSV challenge

[0301] The purpose of this study is to explore whether the duration of immunity of IVP2 will be affected at 6 months after vaccination when used in combination with IVP1. For this purpose, the IVP2 vaccine was reconstituted to the minimum immunizing dose (MID) with IVP1 prepared with high potency. As mentioned above (under A), the combination of IVP1 and IVP2 is also referred to as "IVP2 / IVP1". This study included 70 three-week-old piglets from commercial herds in Germany, of which no animals were antibody or antigen positive to PRRSV when included. The challenge was carried out 182 days after vaccination (six-month duration of immunity). The main parameter of this study was the lung lesions at autopsy 10 to 12 days after the challenge.

[0302] Thus, following PRRSV challenge, animals vaccinated with IVP1 / IVP2 had reduced lung lesions compared to control animals, indicating that the duration of immunity of IVP2 was not negatively affected at 6 months post-vaccination when used in combination with IVP1.

[0303] Embodiment 4:

[0304] Analysis of thiosulfate concentration

[0305] Overview

[0306] As described in Example 1, binary ethyleneimine (BEI) was used as an inactivating agent in the preparation of baculovirus recombinant vaccines. After inactivation with BEI, sodium thiosulfate was added to neutralize the residual BEI.

[0307] After inactivation of the PCV2 antigen fluid with 5mM BEI, small concentrations (usually on the order of about 0.3mM) of BEI are still detectable. The European Pharmacopoeia describes that BEI should not remain in the antigen after inactivation. The European Pharmacopoeia allows the detection of residual sodium thiosulfate after inactivation to be acceptable to ensure that toxic levels of BEI are not retained in the antigen. Therefore, since 5mM BEI is initially added at the beginning of the inactivation step, 5mM thiosulfate is used to neutralize BEI in the activation step. Since a considerable amount of BEI is consumed in the reaction with nucleic acids during inactivation, and since thiosulfate reacts with BEI in a stoichiometric ratio of 1:1, excess residual thiosulfate remains after neutralization.

[0308] The antigenic fluid prepared according to Example 1 was subjected to a thiosulfate test as described below.

[0309] The data from this experiment indicated that the method as described in Example 1 unexpectedly reduced the thiosulfate content in the 10-fold concentrated and diafiltered antigen fluid to below the method detectable limit.

[0310] Materials and Methods

[0311] Quantitative determination of sodium thiosulfate in antigenic fluids by high pressure liquid chromatography (HPLC)

[0312] A. Reagents and Materials

[0313] 1. Sodium thiosulfate pentahydrate, reagent grade of known purity

[0314] 2. Purified water - use 18MΩcm or greater resistivity

[0315] 3. Acetonitrile (ACN), HPLC grade

[0316] 4. Glacial acetic acid, HPLC grade

[0317] 5.Tetrabutylammonium hydroxide (TBA), reagent grade

[0318] 6. HPLC column / protection - Thermo Scientific, ODS-2 Hypersil, 150 x 4.6 mm, 5 micron with appropriate guard, ODS-2 Hypersil-2, 10 x 4.6 mm, 5 micron, or equivalent.

[0319] B. Solution

[0320] 1.20% acetic acid solution

[0321] Combine 10 mL of glacial acetic acid and 40 mL of purified water and mix thoroughly.

[0322] 2. Eluate

[0323] Combine 750 mL of purified water with 20 mL of TBA and mix thoroughly. Adjust the pH of this solution to 4.5-5.0 by adding 20 mL of 20% acetic acid solution and then combine with 250 mL of acetonitrile. Mix thoroughly and filter through a 0.2 μm nylon filter before use.

[0324] 3. Diluent

[0325] Combine 800 mL of purified water and 200 mL of acetonitrile; mix thoroughly and store at ambient conditions.

[0326] C. Thiosulfate Reference Standard Solution

[0327] 1. Sodium thiosulfate stock solution (5mM)

[0328] Weigh 1.2 g of Sodium Thiosulfate Pentahydrate reference material and transfer to a 1000 mL Class A volumetric flask with purified water; mix thoroughly by inversion.

[0329] 2.100% working standard solution (100% WS)

[0330] Transfer 2 mL of the sodium thiosulfate stock solution to a 50 mL Class A volumetric flask. Dilute to volume with diluent and mix thoroughly by inversion.

[0331] 3.50% linear standard solution (50% LS)

[0332] Transfer 1 mL of the sodium thiosulfate stock solution to a 50 mL Class A volumetric flask. Dilute to volume with diluent and mix thoroughly by inversion.

[0333] 4.150% linear standard solution (150% LS)

[0334] Transfer 3 mL of the sodium thiosulfate stock solution to a 50 mL Class A volumetric flask. Dilute to volume with diluent and mix thoroughly by inversion.

[0335] 5. Limit of Quantitation (LOQ)

[0336] Transfer 1 mL of 50% LS to a 20 mL Class A volumetric flask. Dilute to volume with diluent and mix thoroughly by inversion.

[0337] D. PCV2 Inactivated Antigen Fluid Test Solution

[0338] Transfer 500 μL of the inactivated antigen fluid to 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, consuming the first 2 to 3 mL. This is a 20-fold dilution of the antigen fluid.

[0339] E.HPLC Chromatographic Equipment and Conditions

[0340] 1. Flow rate: 1.2mL / min

[0341] 2. Injection volume: 10 μL

[0342] 3. Column: Thermo Scientific, ODS-2 Hypersil, 150 x 4.6 mm, 5 micron, with suitable protection, ODS-2 Hypersil-2, 10 x 4.6 mm, 5 micron.

[0343] 4. Temperature: 20±2℃ sample plate temperature

[0344] 25±2℃ column oven temperature

[0345] 5. Detection: 230nm

[0346] 6. Typical analysis sequence

[0347] ·Dilution agent for waste liquid

[0348] Diluent: 1 injection

[0349] Blank; 1 injection (baseline monitoring, no injection)

[0350] Calibration standard: 6 injections of 100% WS

[0351] Linear standards; 2 injections each of 50, 100 and 150% solution

[0352] LOQ standard; 2 injections

[0353] Test solutions and check standards: 2 injections of each test solution and 2 injections of 100% WS as check standards between the maximum 16 sample injections and the end of the analysis

[0354] 7.Thiosulfate retention time: about 7 minutes.

[0355] F. Calculation of Sodium Thiosulfate Content - Calculate the thiosulfate content (in mM) using the following equation:

[0356] Thiosulfate (mM) = TS / WS × C × 20

[0357] Where: TS - average of the thiosulfate peak areas of two test solution preparation injections.

[0358] WS - average of the thiosulfate area responses for six 100% WS injections.

[0359] C - concentration of working standard solution, expressed in mM (0.2 mM).

[0360] 20 - dilution factor of the test solution

[0361] result

[0362] Several antigen fluids prepared as described in Example 1 were analyzed, wherein the sample pairs obtained were

[0363] (i) After neutralization of BEI with sodium thiosulfate but before downstream processing

[0364] (ii) after downstream processing (i.e., 10-fold concentration and continuous diafiltration) but before mixing with adjuvant and P-saline,

[0365] And determine the concentration of sodium thiosulfate.

[0366] It was found that sample (i) (obtained after BEI neutralization but before downstream processing) still had sodium thiosulfate concentrations in the millimolar range, while unexpectedly no sodium thiosulfate could be detected at or above the detection limit (3 μM) in sample (ii) (obtained after downstream processing but before mixing with adjuvant and P-saline).

[0367] Embodiment 5:

[0368] Analysis of Antigen Fluids by 2D UPLC

[0369] A two-dimensional UPLC method based on anion exchange and size exclusion chromatography separation was used to analyze the antigen containing fluid containing virus-like particles (VLPs) generated by the treatment as described in Example 1.

[0370] The first stage of chromatography used was anion exchange chromatography, and negatively charged species bound to the column (0-2.67 minutes in the chromatogram) and were then eluted by increasing sodium chloride concentration in a gradient.

[0371] The second stage of chromatography used was size exclusion (2.67-15 minutes in the chromatogram) where the valve was switched and the anion exchange from the first dimension was injected onto the size exclusion column. The size exclusion column eluted a mixture of anion exchange eluates.

[0372] Materials and Methods

[0373] A. Reagents and Materials

[0374] 1.1M Tris, pH 7.4, in water, molecular biology grade

[0375] 2.5M NaCl in water, molecular biology grade

[0376] 3. Purified water - use a resistivity of 18M MΩcm or greater

[0377] 4. Anion exchange column - column volume 0.1 mL, with quaternary amine functional groups suitable for binding VLP (macroporous or non-porous material)

[0378] 5. Size Exclusion Column - Column volume 1.75 mL, pore size

[0379] 6.10N NaOH, reagent grade

[0380] B. Solution

[0381] 1.50 mM Tris, pH 8: Combine 50 mL 1 M Tris (pH 7.4) with 900 mL water. Adjust pH to 8 with 10 N NaOH, dilute to 1000 mL, and filter through a 0.22 μm filter.

[0382] 2.50 mM Tris, 2 M NaCl, pH 8: Combine 50 mL 1 M Tris (pH 7.4) and 400 mL 5 M NaCl with 300 mL water. Adjust pH to 8 with 10 N NaOH, dilute to 1000 mL, and filter through a 0.22 μm filter.

[0383] C. PCV2 in Method Reference Solution

[0384] 1. Filter the obtained antigen fluid through a 0.22 μm PES filter before inactivation.

[0385] D. PCV2 Pre-Inactivated or Inactivated Antigen Fluid Test Solution

[0386] 1. Filter the antigen fluid through a 0.22 μm PES filter.

[0387] E.2D-UPLC Chromatographic Equipment and Conditions

[0388] 1. The first dimension of chromatography

[0389] a. Flow rate: 0.6mL / min

[0390] b. Injection volume: 20 μL

[0391] c. System volume: 400 μL

[0392] d. Column: 0.1mL column volume, quadruple amine column

[0393] e. Temperature: Room temperature

[0394] f. Detection: Fluorescence emission (excitation 280nm and emission 330nm)

[0395] g Gradient: A multi-step gradient method with an initial 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 to 10% 50 mM Tris (pH 8) and 90% 50 mM Tris, 2 M NaCl (pH 8) at 2 min was driven through the anion exchange column at a flow rate of 0.6 mL / min.

[0396] 2. The second dimension of the spectrum

[0397] a. Flow rate: 0.3mL / min

[0398] b. Injection from the first dimension: 50 μL

[0399] c. Column: Size exclusion column with a column volume of 1.75 mL and a pore size of

[0400] d. Detection: Fluorescence emission (excitation 280nm and emission 330nm)

[0401] e. Isocratic gradient: 95% 50 mM Tris, pH 8: 5% 50 mM Tris (pH 8), 2 M NaCl

[0402] 3. Typical analysis sequence

[0403] a. Inject 5 2-fold serial dilutions of PCV2 reference solution

[0404] b. Inject 1-15 samples

[0405] c. Inject 5 2-fold serial dilutions of PCV2 reference solution

[0406] 4. Switching time from 1st dimension to 2nd dimension: 2.67 minutes (this switching time depends on the system volume, which is 400 μL in this case)

[0407] 5. The peak ratios listed below were calculated by integrating the peak areas and taking the ratio of the peak areas.

[0408] The VLP (ORF2) product produced by the baculovirus expression system was analyzed using this 2D UPLC. Different prominent peaks can be seen in the resulting chromatogram:

[0409] - The peak at 0-1 min in the chromatogram, also referred to hereinafter as "Peak A", represents components that do not bind to the anion exchange column at pH 8 (i.e., pH 8 neutral or cationic components, such as positively charged proteins and / or cell culture components)

[0410] - The peak at 7-8 minutes in the chromatogram, hereinafter also referred to as "Peak B", represents the VLP (ORF2) antigen of interest

[0411] - The peak at 8.5-9.5 minutes in the chromatogram, hereinafter also referred to as "peak C", represents components in the large size range from IgG (150 kDa) to uracil (0.1 kDa).

[0412] It was found that 10-fold dilutions of samples obtained after downstream processing (i.e., 10-fold concentration and continuous diafiltration) as described in Example 1 showed an average reduction of 99.2% in Peak A and an average reduction of 42% in Peak C compared to upstream processing samples. In addition, while a reduction in Peaks A and C was found, an increase in Peak B was observed.

[0413] Surprisingly, it was found that the peak ratios (Peak A / B and Peak C / B) of the samples obtained after downstream processing as described in Example 1 were only 0.3 (3 replicates, standard deviation 0.1) for Peak A / B and 0.7 (3 replicates, standard deviation 0.2) for Peak C / B.

[0414] It was further found that there was no increase in aggregation or the absence of larger sized species in solution in samples obtained after downstream processing (i.e., 10-fold concentration and sequential diafiltration) as described in Example 1. This observation was surprising, as one skilled in the art would have expected to observe a significant increase in larger species in the concentrated samples on size exclusion chromatography (SEC) due to the dependence of aggregation on concentration.

[0415] Embodiment 6:

[0416] Quantification of free amino acids

[0417] The concentration of amino acids in the antigenic fluid prepared as described in Example 1 was determined by quantifying free amino acids in insect cell culture medium and spent culture medium using conventional methods.

[0418] In brief, the protein in the sample is 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 sample is then derivatized with o-phthalaldehyde (OPA) and mercaptopropionic acid (MPA) in the injection loop of UHPLC, then injected onto a reverse phase column, and quantified by UV absorbance at 338nm. Based on the chromatogram of the standard reference sample (external standard), the concentration of 20 common amino acids (i.e., 20 genetically encoded amino acids) included in the antigen fluid is determined.

[0419] Measuring the amino acid concentrations of several samples taken after downstream processing (i.e., 10-fold concentration and continuous diafiltration) as described in Example 1, and generally considering subsequent dilution of these samples by addition of adjuvant and P-saline, it was found / determined that the characteristic concentrations of specific amino acids in the final product (vaccine) were in the following ranges:

[0420] Tryptophan: 0.1-0.7 μM;

[0421] Glutamine: 0.7-2.4 μM;

[0422] Methionine: 0.5-3.2 μM;

[0423] Arginine: 1.4-4.7 μM;

[0424] Threonine: 1.6-8.0 μM;

[0425] Lysine: 4.0-12.2 μM.

[0426] In this regard, typical average concentrations of these amino acids in the final product (vaccine) were also determined to be within the following ranges:

[0427] Tryptophan: 0.2-0.6 μM;

[0428] Glutamine: 0.8-2.2 μM;

[0429] Methionine: 0.8-2.2 μM;

[0430] Arginine: 1.6-4.3 μM;

[0431] Threonine: 2.4-6.3 μM;

[0432] Lysine: 4.4-11.2 μM.

[0433] In summary, the results obtained from the analyses as described in Examples 4-6 show that the method as described in Example 1 provides a product with specific inherent properties, each of which can contribute to the beneficial results of the testing and studies as described in Examples 2 and 3. BRIEF DESCRIPTION OF THE DRAWINGS

[0434] The following drawings form part of this specification and are included to further illustrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in conjunction with the detailed description of specific embodiments presented herein.

[0435] Figure 1 A schematic overview of an exemplary method according to the invention is provided.

[0436] In the sequence listing:

[0437] SEQ ID NO: 1 corresponds to the sequence of PCV2a ORF2 protein, and

[0438] SEQ ID NO: 2 corresponds to the sequence of the PCV2b ORF2 protein; "PCV2b ORF2 protein" as referred to herein particularly relates to the ORF2 protein known as "mutant PCV2b", which is also considered to belong to genotype d.

[0439] This disclosure contains, among other things, the following items:

[0440] 1. A method for preparing an immunogenic composition comprising a recombinant protein, wherein the method comprises the following steps:

[0441] (a) providing a mixture comprising:

[0442] - a first liquid, and

[0443] - a recombinant protein and / or a quaternary structure comprising a plurality of said recombinant proteins,

[0444] (b) concentrating the recombinant protein and / or the quaternary structure in the mixture produced in step (a) by removing a portion of the first liquid from the mixture, and

[0445] (c) treating the solution resulting from step (b) by continuous diafiltration.

[0446] 2. A method as in item 1, wherein the continuous diafiltration is continuous diafiltration with a second liquid, wherein the second liquid is different from the first liquid.

[0447] 3. A method as in item 1 or 2, wherein the mixture of step (a) additionally contains or further comprises

[0448] - a vector comprising a nucleic acid sequence encoding the recombinant protein, wherein the vector has been inactivated by an inactivating agent, and / or

[0449] - an inactivating agent, which has been neutralized by a neutralizing agent, and / or

[0450] -Neutralizer.

[0451] 4. A method as in item 3, wherein in step (c), the solution produced from step (b) is treated by continuous diafiltration so that the concentration of the neutralized inactivating agent and / or the concentration of the neutralizing agent in the treated solution is reduced.

[0452] 5. A method for preparing an immunogenic composition comprising a recombinant protein, in particular a method according to any one of items 1 to 4, wherein the method comprises the following steps:

[0453] (a)(i) Providing a mixture comprising:

[0454] - a first liquid,

[0455] - a recombinant protein and / or a quaternary structure comprising a plurality of said recombinant proteins, and

[0456] - a vector comprising a nucleic acid sequence encoding the recombinant protein;

[0457] (ii) inactivating the vector by adding an inactivating agent to the mixture of step (i);

[0458] (iii) neutralizing the inactivating agent by adding a neutralizing agent to the mixture resulting from step (ii);

[0459] (b) concentrating the recombinant protein and / or the quaternary structure in the mixture produced in step (a)(iii) by removing a portion of the first liquid from the mixture, and

[0460] (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 in the treated solution is reduced.

[0461] 6. A method as described in any one of items 1 to 5, wherein in step (b), removing a portion of the first liquid from the mixture consists of or comprises filtering the mixture with at least one filter, wherein the at least one filter preferably comprises a filter membrane.

[0462] 7. A method as described in any one of items 3 to 6, wherein in step (b), the concentration comprises

[0463] - feeding said mixture into a filter system comprising at least one filter, wherein said at least one filter comprises a filter membrane having a membrane pore size allowing said neutralized inactivating agent and / or said neutralizing agent to pass therethrough while retaining said recombinant protein and / or said quaternary structure in the bulk flow, and

[0464] - discharging the permeate comprising said neutralized inactivating agent and / or said neutralizing agent.

[0465] 8. A method as described in any one of items 3 to 7, wherein in step (c), the continuous diafiltration comprises

[0466] - feeding said solution into a filter system comprising at least one filter, wherein said at least one filter comprises a filter membrane having a membrane pore size allowing said neutralized inactivating agent and / or said neutralizing agent to pass therethrough while retaining said recombinant protein and / or said quaternary structure in the bulk flow, and

[0467] - discharging a permeate comprising said neutralized inactivating agent and / or said neutralizing agent,

[0468] - adding a second liquid to the bulk flow at a rate equal to the permeate flow, wherein the second liquid is different from the first liquid.

[0469] 9. The method according to any one of items 6 to 8, wherein the at least one filter is at least one flat plate filter and / or at least one hollow fiber filter, and wherein the at least one flat plate filter is preferably at least one cassette filter.

[0470] 10. The method according to any one of items 6 to 9, wherein the at least one filter is 2 to 8 filters, preferably 5 to 7 filters,

[0471] and / or wherein each of the at least one filter has a molecular weight of about 16-26 m 2 , preferably about 20-22m 2 of total filter area.

[0472] 11. A method as described in 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 wherein the filter membrane has a molecular weight cutoff value between about 200 kDa and about 500 kDa.

[0473] 12. The method of any one of items 6 to 11, wherein the filter membrane consists of or comprises a material selected from the group consisting of polyethersulfone, cellulose hydrate, regenerated cellulose, stabilized cellulose, cross-linked cellulose, cross-linked cellulose hydrate, cellulose acetate, polyamide, polyurethane, polypropylene, polysulfone, polycarbonate, nylon, polyimide, and combinations thereof,

[0474] And / or wherein the filter membrane preferably consists of or comprises polyethersulfone, or wherein the filter membrane is optionally a stabilized cellulose based membrane.

[0475] 13. The method according to any one of items 7 to 12, wherein in step (b) and in step (c), the same filter system is used.

[0476] 14. A method according to any one of items 5 to 13, wherein

[0477] - step (iii) is carried out in a first container, and wherein the mixture resulting from the neutralization of the inactivating agent is transferred from the first container to a second container connected to a filter system, and wherein after the mixture is transferred 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,

[0478] - in step (b), circulating the mixture through the second container and the filter system until concentration is complete, and

[0479] - In step (c), the valve between the first container and the second container is opened and the second liquid is continuously directed from the first container to the second container while the mixture circulates through the filter system and the second container.

[0480] 15. The method according to any one of items 1 to 14, wherein the first liquid comprises a part of a cell culture medium or consists of a cell culture medium, and wherein the cell culture medium is preferably an insect cell culture medium.

[0481] 16. A method as described in any one of items 1 to 15, wherein the volume of the mixture produced from step (a) is 1000L to 10000L, preferably 2000L to 8000L, and most preferably 3000L to 5000L.

[0482] 17. A method as described in any one of items 1 to 15, wherein in step (b) the recombinant protein and / or the quaternary structure is ultimately concentrated by at least 5X, preferably at least 8X, more preferably 9X to 11X, compared to the volume of the mixture produced from step (a).

[0483] 18. The method of any one of items 2 to 17, wherein the second liquid is a buffer solution, preferably P-saline or phosphate buffered saline (PBS).

[0484] 19. A method as described in any one of items 8 to 18, wherein in step (c), the total volume of the second liquid added to the overall flow is at least 5X, preferably at least 7X, more preferably at least 9X, the volume of the solution produced in step (b), and / or the total volume of the second liquid added to the overall flow is most preferably approximately the volume of the mixture produced in step (a).

[0485] 20. The method according to any one of items 1 to 19, wherein the method further comprises the following steps:

[0486] (d) mixing the mixture retained after step (c) with a further component selected from a pharmaceutically acceptable carrier, an adjuvant, a diluent, an excipient and a combination thereof, and wherein the concentration of the recombinant protein and / or the concentration of the quaternary structure in the solution resulting from said mixing is preferably about the concentration of the recombinant protein and / or the quaternary structure in the mixture resulting from step (a).

[0487] 21. The method according to any one of items 1 to 20, wherein the mixture used in step (a) is obtainable by a procedure comprising the following steps:

[0488] (1) allowing susceptible cells in culture to be infected with a vector comprising a nucleic acid sequence encoding the recombinant protein, wherein the recombinant protein is expressed by the vector,

[0489] (2) thereafter recovering the recombinant protein and / or the quaternary structure comprising a plurality of the recombinant proteins from the cell culture, wherein cell debris are preferably separated from the recombinant protein and / or the quaternary structure via a separation step, preferably comprising microfiltration via at least one filter, preferably two filters, wherein the at least one filter preferably has a pore size larger than that of the recombinant protein and / or the quaternary structure comprising a plurality of the 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.

[0490] 22. The method of item 21, wherein the separation step comprises or consists of:

[0491] - microfiltration through one or more filters with a pore size of about 2 μm to about 15 μm, and / or

[0492] - Microfiltration through one or more filters having a pore size of about 0.8 μm to about 1.0 μm.

[0493] 23. A method as described in item 21 or 22, wherein the cell culture in step (1) is preferably maintained at 22-32°C when the recombinant protein is expressed by the vector, and / or wherein the recovery in step (2) occurs 5 to 8 days, preferably 8 days, after inoculating the cells with the vector.

[0494] 24. The method of any one of items 1 to 23, wherein the recombinant protein is a PCV2 ORF2 protein, which preferably comprises or preferably 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.

[0495] 25. The method of any one of items 1 to 24, wherein the quaternary structure is a virus-like particle.

[0496] 26. The method according to any one of items 3 to 25, wherein the vector is a recombinant virus, preferably a baculovirus, and / or wherein the nucleic acid sequence is a DNA sequence.

[0497] 27. A method as described in any one of items 3 to 26, wherein the inactivator is an aziridine compound, preferably diethyleneimine (BEI), and / or wherein the inactivator is added in a molar excess relative to the carrier.

[0498] 28. A method as described in any one of items 3 to 27, wherein the neutralizing agent is sodium thiosulfate and / or wherein the neutralizing agent is added in a molar excess relative to the inactivating agent.

[0499] 29. A method as described in any one of items 5 to 28, wherein in step (iii), an equivalent amount of the neutralizing agent is added compared to the amount of inactivating agent added in step (ii).

[0500] 30. A method as described in any one of items 5 to 29, wherein the mixture remaining after step (c) contains a fire extinguishing agent concentration that is less than one thousandth of the concentration of the neutralizing agent produced from step (iii).

[0501] 31. A method as described in any one of items 1 to 30, wherein the virucidal activity of the immunogenic composition produced by the method is reduced by at least 20% compared to an immunogenic composition mixture that has not undergone the concentration of step (b) and the continuous diafiltration of step (c) of the method, and / or wherein when live virus is mixed with the immunogenic composition prepared by the method for 4 hours or more, in particular for 4 hours or more at room temperature, the immunogenic composition results in less than 1 log TCID 50 / mL of live virus, preferably less than 0.7 log TCID 50 / mL loss of live virus.

[0502] 32. A method as described in any one of items 1 to 31, wherein the method further comprises the step of combining the mixture retained after step (c) and / or step (d) with at least one additional antigen.

[0503] 33. The method of item 31 or 32, wherein the live virus or the at least one additional antigen is porcine reproductive and respiratory syndrome (PRRS) virus.

[0504] 34. An immunogenic composition obtainable by the method of any one of items 1 to 33.

[0505] 35. The immunogenic composition of item 34, wherein the immunogenic composition is obtainable by the method of any one of items 28 to 33, and wherein the mixture remaining after step (c) contains less than 3 μM sodium thiosulfate.

[0506] 36. The immunogenic composition of item 34, wherein the immunogenic composition is obtainable by the method of any one of items 28 to 33, and wherein the immunogenic composition produced from step (d) is substantially free of sodium thiosulfate.

[0507] 37. An immunogenic composition, in particular the immunogenic composition of any one of items 34 to 36, comprising:

[0508] - 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 to the sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2, and

[0509] - 1-10 μM L-arginine and / or 1-10 μM L-lysine,

[0510] And wherein preferably, the immunogenic composition is substantially free of neutralized inactivating agent and / or substantially free of neutralizing agent.

[0511] 38. An immunogenic composition, in particular an immunogenic composition according to any one of items 34 to 37, preferably obtainable by any of the methods comprising step (d), wherein the immunogenic composition comprises

[0512] 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 to the sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2,

[0513] and

[0514] 0.1-1 μM L-tryptophan; and / or

[0515] 0.1-3 μM L-glutamine; and / or

[0516] 0.2-4 μM L-methionine; and / or

[0517] 1-10 μM L-arginine; and / or

[0518] 1-10 μM L-threonine; and / or

[0519] 1-15 μM L-lysine.

[0520] 39. The immunogenic composition of any one of items 34 to 38, wherein the immunogenic composition comprises

[0521] 0.1-0.7 μM L-tryptophan; and / or

[0522] 0.7-2.4 μM L-glutamine; and / or

[0523] 0.5-3.2 μM L-methionine; and / or

[0524] 1.4-4.7 μM L-arginine; and / or

[0525] 1.6-8.0 μM L-threonine; and / or

[0526] 4.0-12.2 μM L-lysine.

[0527] 40. The immunogenic composition of claim 39, wherein the immunogenic composition comprises

[0528] 0.2-0.6 μM L-tryptophan; and / or

[0529] 0.8-2.2 μM L-glutamine; and / or

[0530] 0.8-2.2 μM L-methionine; and / or

[0531] 1.6-4.3 μM L-arginine; and / or

[0532] 2.4-6.3 μM L-threonine; and / or

[0533] 4.4-11.2 μM L-lysine.

[0534] 41. The immunogenic composition of any one of items 34 to 40, wherein the composition comprises

[0535] 0.7-2.4 μM L-glutamine and 4.0-12.2 μM L-lysine.

[0536] 42. The immunogenic composition of any one of items 34 to 41, wherein the composition comprises

[0537] 0.8-2.2 μM L-glutamine and 4.4-11.2 μM L-lysine.

[0538] 43. An immunogenic composition, in particular the immunogenic composition of any one of items 34 to 42, preferably obtainable by any of the methods comprising step (d), wherein the immunogenic composition exhibits a ratio of Peak A area to Peak B area of ​​0.2-0.4 and / or a ratio of Peak C area to Peak B area of ​​0.5-0.9 when subjected to two-dimensional (2D) ultra performance liquid chromatography (UPLC) at room temperature, wherein the 2D UPLC comprises

[0539] The first dimension of chromatography had a system volume of 400 μL, where

[0540] - injecting a sample comprising said immunogenic composition into an anion exchange column wherein 0.1 mL of the column volume is filled with a quaternary amine functionalized material suitable for binding virus-like particles, and

[0541] - a multi-step gradient method driving initially 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 to 10% 50 mM Tris (pH 8) and 90% 50 mM Tris, 2 M NaCl (pH 8) at 2 min at a flow rate of 0.6 mL / min through the anion exchange column,

[0542] and wherein at a retention time of 2.67 min, it switches from the first dimension of the chromatography to

[0543] The second dimension of chromatography, wherein 50 μL of the eluate from the first dimension of chromatography was driven at a flow rate of 0.3 mL / min through a column having a volume of 1.75 mL and A size exclusion column with a pore size of

[0544] and wherein the chromatogram is recorded by monitoring the fluorescence emission at a wavelength of 330 nm and an excitation wavelength of 280 nm, and wherein

[0545] Peak A area is the peak area of ​​the highest peak with a retention time of 0-1 minute in the chromatogram;

[0546] Peak B area is the peak area of ​​the highest peak with a retention time of 7-8 minutes in the chromatogram;

[0547] The peak C area is the peak area of ​​the highest peak at a retention time of 8.5-9.5 minutes in the chromatogram.

[0548] 44. The immunogenic composition of any one of items 34 to 43, wherein the immunogenic composition is substantially free of neutralizing agents, preferably substantially free of sodium thiosulfate.

[0549] 45. The immunogenic composition of any one of items 34 to 44, wherein the immunogenic composition further comprises an attenuated live virus, preferably an attenuated porcine reproductive and respiratory syndrome (PRRS) virus or an attenuated live bacteria.

[0550] 46. ​​The immunogenic composition of any one of items 34 to 45, wherein after administration of one dose of the immunogenic composition, the immunogenic composition induces a protective immune response against the pathogen from which the amino acid sequence of the recombinant protein is derived, preferably against PCV2.

[0551] 47. The immunogenic composition of any one of items 45 or 46, wherein the immunogenic composition induces a protective immune response against PRRS virus following administration of one dose of the immunogenic composition.

[0552] 48. A kit comprising a container containing the immunogenic composition of any one of items 34 to 47.

[0553] 49. A kit comprising a container containing an immunogenic composition prepared by the method of any one of items 1 to 33.

[0554] 50. The kit of claim 48 or 49, further comprising at least one additional container containing at least one additional antigen selected from the group consisting of attenuated live viruses, preferably attenuated PRRS viruses, and attenuated live bacteria.

[0555] 51. The immunogenic composition of any one of items 34 to 50 and / or the kit of any one of items 48 to 50, further comprising one or more attenuated or inactivated non-PRRSV pathogens or antigenic substances thereof.

[0556] 52. The immunogenic composition according to any one of items 34 to 47 and 51 and / or the kit according to any one of items 48 to 50 for use as a medicament, preferably as a vaccine.

[0557] 53. The immunogenic composition of any one of items 34 to 47 and 51 to 52 and / or the kit of any one of items 48 to 52 for use in an animal in the following method

[0558] - Induce a protective immune response against at least one pathogen, and / or

[0559] - Reduction in one or more clinical signs of infection with at least one pathogen

[0560] The at least one pathogen is preferably selected from the group consisting of PCV2, PRRSV, non-PRRSV pathogens and combinations thereof.

[0561] 54. The immunogenic composition and / or kit according to item 51, in particular for use according to item 52 or 53, wherein the non-PRRSV pathogen is selected from Mycoplasma hyopneumoniae, pseudorabies virus, swine influenza virus, porcine parvovirus, Transmissibie gastroenteritis virus, Escherichia coli, Erysipelothrix rhizopus, Bordetella bronchiseptica, Salmonella choleraesuis, Haemophilus parasuis, Pasteurella multocida, Streptococcus suis and Actinobacillus pleuropneumoniae.

[0562] 55. A method for producing an immunogenic composition comprising a recombinant protein, wherein the method comprises the following steps:

[0563] (a) providing a mixture comprising:

[0564] - a first liquid, and

[0565] - a recombinant PCV2 ORF2 protein and / or a virus-like particle comprising a plurality of recombinant PCV2 ORF2 proteins,

[0566] (b) concentrating the recombinant PCV2 ORF2 protein and / or the virus-like particles in the mixture produced in step (a) by removing a portion of the first liquid from the mixture, thereby forming a bulk solution, and

[0567] (c) treating the bulk solution resulting from step (b) by continuous diafiltration to form a treated solution.

[0568] 56. A method as in item 55, wherein the mixture of step (a) additionally contains or further comprises

[0569] - a vector comprising a nucleic acid sequence encoding the recombinant PCV2 ORF2 protein, wherein the vector has been inactivated by an inactivating agent, and / or

[0570] - an inactivating agent, which has been neutralized by a neutralizing agent, and / or

[0571] - neutralizing agent,

[0572] and / or wherein in step (c) the bulk effluent solution resulting from step (b) is treated by continuous diafiltration such that the concentration of neutralised inactivating agent and / or the concentration of neutralising agent in the treated solution is reduced.

[0573] 57. The method of item 55 or 56, wherein the method comprises the following steps:

[0574] (a)(i) Providing a mixture comprising:

[0575] - a first liquid,

[0576] - recombinant PCV2 ORF2 protein and / or virus-like particles comprising a plurality of recombinant PCV2 ORF2 proteins, and

[0577] - a vector comprising a nucleic acid sequence encoding the recombinant protein;

[0578] (ii) inactivating the vector by adding an inactivating agent to the mixture of step (i);

[0579] (iii) neutralizing the inactivating agent by adding a neutralizing agent to the mixture resulting from step (ii);

[0580] (b) concentrating the recombinant PCV2 ORF2 protein and / or the virus-like particles in the mixture produced in step (a)(iii) by removing a portion of the first liquid from the mixture, thereby forming a bulk solution, and

[0581] (c) treating the bulk effluent solution resulting from step (b) by continuous diafiltration to form a treated solution such that the concentration of neutralized inactivating agent and / or the concentration of neutralizing agent in the treated solution is reduced.

[0582] 58. A method as described in any one of items 55 to 57, 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, wherein the at least one filter preferably comprises a filter membrane.

[0583] 59. The method of any one of items 55 to 58, wherein the mixture of step (a) is concentrated by:

[0584] - forming a permeate by feeding said mixture to a filter system comprising at least one filter, wherein said at least one filter comprises a filter membrane having a membrane pore size that allows said neutralized inactivating agent and / or said neutralizing agent to pass therethrough while retaining said recombinant PCV2 ORF2 protein and / or said virus-like particles in the bulk flow solution, and

[0585] - wherein the permeate comprises the neutralized inactivating agent and / or the neutralizing agent,

[0586] - discharge of said permeate,

[0587] and / or wherein the continuous diafiltration of step (c) comprises

[0588] - forming a permeate by feeding the bulk flow solution of step (b) to a filter system comprising at least one filter, wherein the at least one filter comprises a filter membrane having a membrane pore size that allows the neutralized inactivating agent and / or the neutralizing agent to pass therethrough while retaining the recombinant PCV2 ORF2 protein and / or the virus-like particles in the bulk flow solution, and

[0589] - wherein the permeate comprises the neutralized inactivating agent and / or the neutralizing agent,

[0590] - discharging said permeate, and

[0591] - adding a second liquid to the bulk flow solution at a rate equal to the rate at which the permeate is discharged,

[0592] The second liquid is different from the first liquid.

[0593] 60. The method of item 58 or 59, wherein the at least one filter is at least one flat plate filter and / or at least one hollow fiber filter, and wherein the at least one flat plate filter is preferably at least one cassette filter,

[0594] and / or wherein the at least one filter is 2-8 filters, preferably 5-7 filters,

[0595] and / or wherein each of the at least one filter has a molecular weight of about 16-26 m 2 , preferably about 20-22m 2 The total filter area,

[0596] and / or wherein the filter membrane has an average pore size smaller than the recombinant PCV2 ORF2 protein and / or the virus-like particle, and / or wherein the filter membrane has a molecular weight cutoff between about 200 kDa and about 500 kDa,

[0597] and / or wherein the filter membrane consists of or comprises a material selected from the group consisting of polyethersulfone, cellulose hydrate, regenerated cellulose, stabilized cellulose, cross-linked cellulose, cross-linked cellulose hydrate, cellulose acetate, polyamide, polyurethane, polypropylene, polysulfone, polycarbonate, nylon, polyimide, and combinations thereof,

[0598] And / or wherein the filter membrane preferably consists of or comprises polyethersulfone, or wherein the filter membrane is optionally a stabilized cellulose based membrane.

[0599] 61. The method of item 59 or 60, wherein in step (b) and in step (c), the same filter system is used.

[0600] 62. A method according to any one of items 57 to 61, wherein

[0601] - step (iii) is carried out in a first container, and wherein the mixture of step (a) resulting from the neutralization of the inactivating agent is transferred from the first container to a second container connected to a filter system, and wherein after the mixture of step (a) is transferred 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,

[0602] - in step (b), circulating the mixture of step (a) through the second container and the filter system until the mixture of step (a) is concentrated to form the bulk solution of step (b); and

[0603] - In step (c), the valve between the first container and the second container is opened and the second liquid is continuously directed from the first container to the second container while the bulk flow solution circulates through the filter system and the second container until a treatment solution is formed.

[0604] 63. The method according to any one of items 55 to 62, wherein the first liquid comprises part of a cell culture medium or consists of a cell culture medium, and wherein the cell culture medium is preferably an insect cell culture medium,

[0605] and / or wherein the volume of the mixture resulting from step (a) is from 1000 L to 10000 L, preferably from 2000 L to 8000 L, and most preferably from 3000 L to 5000 L,

[0606] and / or wherein in step (b), the mixture of step (a) is concentrated into a bulk flow solution until the bulk flow solution is concentrated by at least 5X, preferably at least 8X, more preferably 9X to 11X in recombinant PCV2 ORF2 protein and / or said virus-like particles compared to the concentration of recombinant PCV2 ORF2 protein and / or said virus-like particles in the volume of said mixture resulting from step (a).

[0607] 64. A method as described in any one of items 59 to 63, wherein the second liquid is a buffer solution, preferably P-saline or phosphate buffered saline (PBS),

[0608] and / or wherein in step (c), the total volume of the second liquid added to the bulk flow solution is at least 5X, preferably at least 7X, more preferably at least 9X, of the volume of the bulk flow solution resulting from step (b), and / or the total volume of the second liquid added to the bulk flow solution is most preferably approximately the volume of the concentrated mixture resulting from step (a).

[0609] 65. The method of any one of items 55 to 64, wherein the method further comprises the following steps:

[0610] (d) forming a solution D by mixing the treatment solution retained after step (c) with a further component selected from a pharmaceutically acceptable carrier, an adjuvant, a diluent, an excipient and a combination thereof, and wherein the concentration of the recombinant PCV2 ORF2 protein and / or the concentration of the virus-like particles in the solution D produced from said mixing is preferably about the concentration of the PCV2 ORF2 recombinant protein and / or the virus-like particles in the mixture produced from step (a).

[0611] 66. The method of any one of items 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.

[0612] 67. The method according to any one of items 56 to 66, wherein the vector is a recombinant virus, preferably a baculovirus, and / or wherein the nucleic acid sequence is a DNA sequence,

[0613] and / or wherein the inactivator is an aziridine compound, preferably diethyleneimine (BEI), and / or wherein the inactivator is added in a molar excess relative to the carrier,

[0614] and / or wherein the neutralizing agent is sodium thiosulfate and / or wherein the neutralizing agent is added in molar excess relative to the inactivating agent.

[0615] 68. A method as described in any one of items 55 to 67, wherein the immunogenic composition in solution D has virucidal activity, and wherein the virucidal activity of the immunogenic composition produced by the method 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 the method, and / or wherein when live virus is mixed with the immunogenic composition produced by the method at room temperature for 4 hours or more, the immunogenic composition results in less than 1 log TCID 50 / mL of live virus, preferably less than 0.7 log TCID 50 / mL loss of live virus.

[0616] 69. The method of claim 68, wherein the live virus is mixed with the immunogenic composition at room temperature for 4 hours or more.

[0617] 70. The method of any one of items 55 to 69, wherein the method further comprises the step of combining the treatment solution retained after step (c) and / or the solution D retained after step (d) with at least one additional antigen,

[0618] And / or wherein the at least one additional antigen is Porcine Reproductive and Respiratory Syndrome (PRRS) virus.

[0619] 71. An immunogenic composition obtainable by the method of any one of items 55 to 70.

[0620] 70. The immunogenic composition of claim 71, comprising:

[0621] - 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 to the sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2, and

[0622] - 1-10 μM L-arginine and / or 1-10 μM L-lysine,

[0623] And wherein preferably, the immunogenic composition is substantially free of neutralized inactivating agent and / or substantially free of neutralizing agent.

[0624] 73. The immunogenic composition of item 71 or 72, wherein the immunogenic composition further comprises an attenuated live virus, preferably an attenuated porcine reproductive and respiratory syndrome (PRRS) virus or an attenuated live bacterium,

[0625] and / or wherein upon administration of one dose of said immunogenic composition, said immunogenic composition induces a protective immune response against PCV2,

[0626] And / or wherein the immunogenic composition induces a protective immune response against PRRS virus following administration of one dose of the immunogenic composition.

[0627] 74. A kit comprising a container containing the immunogenic composition of any one of items 71 to 73, and wherein the kit preferably comprises 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.

[0628] 75. The immunogenic composition according to any one of items 71 to 73 and / or the kit according to item 74 for use as a medicament, preferably as a vaccine.

[0629] 76. The immunogenic composition of any one of items 71 to 73 and 75 and / or the kit of items 74 or 75, for use in an animal in the following method

[0630] - Induce a protective immune response against at least one pathogen, and / or

[0631] - Reduction in one or more clinical signs of infection with at least one pathogen

[0632] The at least one pathogen is preferably selected from the group consisting of PCV2, PRRSV, non-PRRSV pathogens and combinations thereof.

Claims

1. A method for preparing an immunogenic composition comprising a recombinant protein, wherein the method comprises the following steps: (a) providing a mixture comprising: - a first liquid, and - a recombinant PCV2 ORF2 protein and / or a virus-like particle comprising a plurality of recombinant PCV2 ORF2 proteins, (b) concentrating the recombinant PCV2 ORF2 protein and / or the virus-like particles in the mixture produced in 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.

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

3. A method for preparing an immunogenic composition comprising a recombinant PCV2 ORF2 protein, in particular the method of claim 2, wherein the method comprises the following steps: (a)(i) Providing a mixture comprising: - a first liquid, - recombinant PCV2 ORF2 protein and / or virus-like particles comprising a plurality of recombinant PCV2 ORF2 proteins, and - a vector comprising a nucleic acid sequence encoding the recombinant protein; (ii) inactivating the vector by adding an inactivating agent to the mixture of step (i); (iii) neutralizing the inactivating 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 produced in 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 in the treated solution is reduced.

4. The method of 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, wherein the at least one filter preferably comprises a filter membrane.

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

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

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

8. The method of any one of claims 3 to 7, wherein - step (iii) is carried out in a first container, and wherein the mixture resulting from the neutralization of the inactivating agent is transferred from the first container to a second container connected to a filter system, and wherein after the mixture is transferred 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), circulating the mixture through the second container and the filter system until concentration is complete, and - In step (c), the valve between the first container and the second container is opened and the second liquid is continuously directed from the first container to the second container while the mixture circulates through the filter system and the second container.

9. The method according to any one of claims 1 to 8, wherein the first liquid comprises a part of a cell culture medium or consists of a cell culture medium, and wherein the cell culture medium is preferably an insect cell culture medium, and / or wherein the volume of the mixture resulting from step (a) is from 1000 L to 10000 L, preferably from 2000 L to 8000 L, and most preferably from 3000 L to 5000 L, And / or wherein in step (b), the recombinant PCV2 ORF2 protein and / or the virus-like particles are ultimately concentrated by at least 5X, preferably at least 8X, more preferably 9X to 11X, compared to the volume of the mixture produced from step (a).

10. The method of any one of claims 5 to 9, wherein 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 flow is at least 5X, preferably at least 7X, more preferably at least 9X, the volume of the solution resulting from step (b), and / or wherein the total volume of the second liquid added to the bulk flow is most preferably approximately the volume of the mixture resulting from step (a).

11. The method of any one of claims 1 to 10, wherein the method further comprises the following steps: (d) mixing the mixture remaining after step (c) with a further component selected from a pharmaceutically acceptable carrier, an adjuvant, a diluent, an excipient and a combination thereof, and wherein the concentration of the recombinant PCV2 ORF2 protein and / or the concentration of the virus-like particle structure in the solution resulting from said mixing is preferably about the concentration of the PCV2 ORF2 recombinant protein and / or the virus-like particle structure in the mixture resulting from step (a).

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 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.

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

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

15. The method of any one of claims 1 to 14, wherein the method further comprises the step of combining the mixture remaining after step (c) and / or step (d) with at least one additional antigen, And / or wherein the at least one additional antigen is preferably Porcine Reproductive and Respiratory Syndrome (PRRS) virus.

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

17. An immunogenic composition, in particular the immunogenic composition of claim 16, comprising: - a recombinant PCV2 ORF2 protein, which preferably comprises or preferably 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, and - 1-10 μM L-arginine and / or 1-10 μM L-lysine, And wherein preferably, the immunogenic composition is substantially free of neutralized inactivating agent and / or substantially free of neutralizing agent.

18. The immunogenic composition of claim 16 or 17, wherein the immunogenic composition further comprises an attenuated live virus, preferably an attenuated porcine reproductive and respiratory syndrome (PRRS) virus or an attenuated live bacterium, and / or wherein upon administration of one dose of said immunogenic composition, said immunogenic composition induces a protective immune response against PCV2, And / or wherein the immunogenic composition induces a protective immune response against PRRS virus following administration of one dose of the immunogenic composition.

19. A kit comprising a container containing the immunogenic composition of any one of claims 16 to 18, and wherein the kit preferably comprises 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.

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

21. The immunogenic composition of any one of claims 16 to 18 and 20 and / or the kit of claim 19 or 20 for use in an animal in the following method - Induce a protective immune response against at least one pathogen, and / or - Reduction in one or more clinical signs of infection with at least one pathogen The at least one pathogen is preferably selected from the group consisting of PCV2, PRRSV, non-PRRSV pathogens and combinations thereof.

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