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New emerging technologies delivering benefits in terms of process robustness and economy are an inevitable prerequisite for monoclonal antibody purification processes intensification. Caprylic acid was proven as an effective precipitating agent enabling efficient precipitaton of product- and process-related impurities while leaving the antibody in solution. This purification step at mild acidic pH was therefore introduced in generic antibody platform approaches after Protein A capture and evaluated for its impact regarding process robustness and antibody stability. Comparison of 13 different monoclonal antibodies showed significant differences in antibody recovery between 65-95% during caprylic acid-induced impurity precipitation. Among six compared physicochemical properties, isoelectric point of the antibody domains was figured out to correlate with yield. Antibodies with mild acidic pI of the light chain were significantly susceptible to caprylic acid-induced precipitation resulting in lower yields. Virus clearance studies revealed that caprylic acid provided complete virus inactivation of an enveloped virus. Multiple process relevant factors such as pH range, caprylic acid concentration and antibody stability were investigated in this study to enable an intensified purification process including caprylic acid precipitation for HCP removal of up to 2 log10 reduction values at mAb yields >90% while also contributing to the virus safety of the process.

Chromatofocusing was performed in order to separate a polyclonal antigen-specific mixture of human immunoglobulins (IgGs) that would then allow for further analyses of as few different IgGs as possible. Because polyclonal IgGs only differ by amino acid sequence and possible post-translational modifications but not by molecular weight, we chose chromatofocusing for protein separation by different isoelectric points. We isolated antigen-specific IgGs from commercially available intravenous immunoglobulins (IVIG) using a combination of affinity- and size exclusion-chromatography and in order to reduce the complexity of the starting material IVIG was then replaced by single-donor plasmapheresis material. Using two-dimensional gel electrophoresis (2-DE), we observed a clear decrease in the number of different light and heavy chains in the chromatofocusing peak as compared to the starting material. In parallel, we monitored slight problems with the selected peak in isoelectric focusing as the first dimension of 2-DE, displayed in by the less proper focusing of the spots. When we tested whether IgGs were binding to their specific antigen after chromatofocusing, we were able to show that they were still in native conformation. In conclusion, we showed that chromatofocusing can be used as a first step in the analysis of mixtures of very similar proteins, e.g. polyclonal IgG preparations, in order to minimize the amount of different proteins in separated fractions in a reproducible way.

Dynamic binding capacity (DBC) measurements of cation-exchange resins were performed with two human monoclonal antibodies. DBC showed a pH dependent maximum, which was shifted to lower pH values with increasing buffer concentrations and increasing salting-out effect of the buffer anion according to the Hofmeister series. As this downshift correlates well with zeta potential values, a measurement of the latter allows the determination of the pH value for maximum DBC under a given set of conditions. Thus, the use of zeta potential values can accelerate the purification process development and helps to understand the protein adsorption mechanism.

Die biotechnologische Herstellung therapeutischer Proteine ist ein aufwendiger und streng regulierter Prozess. Die richtige Formulierung und deren Entwicklung ist eine grundlegende Voraussetzung zur Gewahrleistung der Qualitat und des Erfolgs eines biopharmazeutischen Produktes.

The pH dependence in hydrophobic interaction chromatography (HIC) is usually discussed exclusively in terms of protein dependence and there are no clear defined trends. Many of the deviations from an ideal solution are caused solely by the high salt concentration, as protein concentration is usually negligible. So pH dependency in hydrophobic interaction chromatography could also be the result of pH dependent changes of ion properties from the salt solution. The possibility that pH dependent ion hydration or ion association in highly concentrated salt solutions may influence the dynamic protein binding capacity onto HIC resins was investigated. In buffer solutions commonly used in HIC e.g. sodium chloride, ammonium sulphate and sodium citrate pH dependent maxima in the electro-acoustic signals were found. These maxima are related to an increase of the ion sizes by hydration or ion association. At low ionic strength the maxima are in the range between 4.5 and 6 and they increased in concentrated electrolyte solutions to values between 6 and 8. The range of these maxima is in the same region as dynamic protein binding capacity maxima often observed in HIC. For a qualitative interpretation of this phenomenon of increased protein stabilization by volume exclusion effect extended scaling theory can be used. This theory predicts a maximum of protein stabilization if the ratio of salt ion diameter to water is 1.8. According to the hypothesis raised here, if the pH dependent ratio of salt ion diameter to water approaches this value the transport of the protein in the pore system is less restricted and an increase in binding capacity can be produced.