An inert tracer: The binding site of a fluorescent dye on the antibody and its effects on Protein A chromatography.

Fluorescently labeled antibodies are widely used to visualize the adsorption process in protein chromatography using confocal laser scanning microscopy (CLSM), but also as a tracer for determination of residence time distribution (RTD) in continuous chromatography. It is assumed that the labeled protein is inert and representative of the unlabeled antibody, ignoring the fact that labeling with a fluorescent dye can change the characteristics of the original molecule. It became evident that the fluorescently labeled antibody has a higher affinity toward protein A resins such as MabSelect Sure. This can be due to slight differences in hydrophobicity and net charge, which are caused by the addition of the fluorescent dye. However, this difference is eliminated when using high salt concentrations in the adsorption studies. In this work, the site occupancy of two labeled antibodies, MAb1 (IgG1 subclass) and MAb2 (IgG2 subclass) conjugated with the fluorescent dye Alexa Fluor™ 488 was elucidated by intact mass spectrometry (MS) and peptide mapping LC-MS/MS, employing a sequential cleavage with Endoproteinase Lys-C and trypsin and in parallel with chymotrypsin alone. It was shown that the main binding site for the dye was a specific lysine in the heavy chains of the MAb1 and MAb2 molecules, in positions 188 and 189 respectively. Other lysine residues distributed throughout the protein sequence were labeled to a lot lesser extent. The labeled antibody had a slightly different affinity to MabSelect Sure although its primary binding site (to Protein A) was not affected by labeling, despite the secondary region responsible for binding to the protein A was partly labeled. Overall, the fluorescent-labeled antibodies are a good compromise as an inert tracer in residence time distribution and chromatography studies because they are much cheaper than isotope-labeled antibodies; However, the differences between the labeled and unlabeled antibodies should be considered.

Cytokines as fast indicator of infectious virus titer during process development.

Measuring infectious titer is the most time-consuming method during the production and process development of live viruses. Conventionally, it is done by measuring the tissue culture infectious dose (TCID50) or plaque forming units (pfu) in cell-based assays. Such assays require a time span of more than a week to the readout and significantly slow down process development. In this study, we utilized the pro-inflammatory cytokine response of a Vero production cell line to a recombinant measles vaccine virus (MVV) as model system for rapidly determining infectious virus titer within several hours after infection instead of one week. Cytokines are immunostimulatory proteins contributing to the first line of defence against virus infection. The probed cytokines in this study were MCP-1 and RANTES, which are secreted in a virus dose as well as time dependent manner and correlate to TCID50 over a concentration range of several logarithmic levels with R2 = 0.86 and R2 = 0.83, respectively. Furthermore, the pro-inflammatory cytokine response of the cells was specific for infectious virus particles and not evoked with filtered virus seed. We also discovered that individual cytokine candidates may be more suitable for off- or at-line analysis, depending on the secretion profile as well as their sensitivity towards changing process conditions. Furthermore, the method can be applied to follow a purification procedure and is therefore suited for process development and control.

Residence time distribution in counter-current protein A affinity chromatography using an inert tracer.

The trend in the biopharmaceutical industry is changing from batch process to continuous process. For continuous biomanufacturing, traceability of the material is required by regulatory authorities. The recent ICH draft guideline Q13 on continuous manufacturing of drug substances and drug products requests an "understanding of process dynamics as a function of input material attributes (e.g., potency, material flow properties), process conditions (e.g., mass flow rates) … One common approach is characterization of residence time distribution (RTD) for the individual unit operations and integrated system." Thus, it is necessary to trace material through individual continuous unit operations and the integrated process. The RTD of a process is obtained experimentally by injecting a pulse of an inert tracer into the inlet and measuring the broadening of the injected pulse in the outlet. We investigated the RTD of three-column periodic counter-current chromatography (PCC) using staphylococcal protein A affinity chromatography, with a focus on how the material distributes over subsequent cycles. A fluorescent-labeled antibody was used as the inert tracer under high salt concentration. The tracer was injected once in each run but at different points of the loading phase. We then analyzed the outlet of the column. In the elution phase, regardless of the point of injection, we observed an even distribution of the tracer. In the loading phase, a constant exchange between the antibody in the solid phase and the liquid phase was observed, meaning that sending the outlet of one chromatography column into another column to improve resin utilization causes higher residence time in the system for some portion of the material.