Systems and methods for performing a real-time assay of a sample

A closed system for performing an online, real-time assay, the system comprising:

• a first holding coil fluidly arranged to receive a sample of a product containing polypeptides;
• a multi-port valve fluidly coupled to and located downstream of the first holding coil;
• a polypeptide binding column fluidly coupled to the multi-port valve and arranged to receive the sample from the first holding coil via a first port of the multi-port valve, the polypeptide binding column configured to bind the polypeptides from the sample,
• a buffer source fluidly coupled to the multi-port valve and arranged to supply elution buffer solution to a second holding coil located downstream of the polypeptide binding column, such that the elution buffer solution is adapted to elute substantially all of the polypeptides from the polypeptide binding column;
• a reaction chamber fluidly coupled to the multi-port valve and arranged downstream of the polypeptide binding column, the reaction chamber adapted to receive a mixture from the polypeptide binding column via a second port of the multi-port valve, the mixture comprising the elution buffer solution and the eluted polypeptides, wherein the polypeptides of the mixture are denatured in the reaction chamber, wherein the reaction chamber is arranged to receive a reducing reagent that cleaves disulfide bond crosslinks via the first holding coil and a third port of the multi-port valve, the reducing reagent reduces the denatured polypeptides, and wherein the reaction chamber is further arranged to receive an alkylating reagent that alkylates sulfhydryls via the first holding coil and the third port of the multi-port valve, wherein the alkylating reagent alkylates the denatured and reduced polypeptides in the reaction chamber;
• a desalting column fluidly coupled to the multi-port valve and arranged to receive the denatured, reduced, and alkylated polypeptides, the elution buffer solution, and the alkylating reagent from the reaction chamber, the desalting column configured to separate the denatured, reduced, and alkylated polypeptides from the elution buffer solution, the reducing reagent, and the alkylating reagent;
• and
• a proteolytic enzyme column fluidly coupled to and arranged downstream of the second polypeptide column to obtain the separated polypeptides from the desalting column, the proteolytic enzyme column configured to digest the desalted polypeptides.