Method of producing an immunogenic composition

A method of producing an immunogenic composition comprising a recombinant PCV2 ORF2 protein, wherein the method comprises the steps of:

• (a)(i) providing a mixture containing
• a first liquid,
• recombinant PCV2 ORF2 protein and/or virus-like particles comprising a plurality of a recombinant PCV2 ORF2 protein, and
• a vector comprising a nucleic acid sequence encoding said 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), wherein the neutralization is carried out in a first container, and wherein the mixture resulting from neutralizing the inactivating agent is transferred from the first container to a second container connected with a filter system, and wherein after transferring the mixture from the first to the second container a valve between the first container and the second container is closed, and the empty first container is filled with a second liquid different from the first liquid;
• (b) concentrating the recombinant PCV2 ORF2 protein and/or said virus-like particles in the mixture resulting from step (a)(iii) by removing a portion of the first liquid from the mixture by circulating the mixture through the second container and the filter system until the concentrating is completed, and
• (c) processing the solution resulting from step (b) by continuous diafiltration such that the concentration of the neutralized inactivating agent and/or the concentration of the neutralizing agent is decreased in the process solution, wherein said continuous diafiltration comprises:
• feeding the solution into the filter system containing at least one filter, wherein the at least one filter comprises a filter membrane having a membrane pore size allowing the neutralized inactivating agent and/or the neutralizing agent to pass through while retaining the recombinant PCV2 ORF2 protein and/or said virus-like particles in the bulk flow,
• discharging the permeate comprising the neutralized inactivating agent and/or the neutralizing agent,
• adding the second liquid to the bulk flow at a rate equal to the permeate flow, wherein the valve between the first and the second container is opened and the second liquid is continuously led from the first container to the second container while the mixture is circulated through the filter system and the second container.