Variable tangential flow filtration

Abstract

The current invention reports a method for concentrating an immunoglobulin solution by tangential flow filtration wherein the transmembrane pressure and the cross-flow are variable.

Description

[0001]The current invention is in the field of protein concentration, to be more precise it relates to the use of tangential flow filtration (TFF) for immunoglobulin concentration.BACKGROUND OF THE INVENTION[0002]Proteins and especially immunoglobulins play an important role in today's medical portfolio. Expression systems for the production of recombinant polypeptides are well-known in the state of the art and are described by, e.g., Marino, M. H., Biopharm. 2 (1989) 18-33; Goeddel, D. V., et al., Methods Enzymol. 185 (1990) 3-7; Wurm, F., and Bernard, A., Curr. Opin. Biotechnol. 10 (1999) 156-159. Polypeptides for use in pharmaceutical applications are mainly produced in mammalian cells such as CHO cells, NSO cells, Sp2 / 0 cells, COS cells, HEK cells, BHK cells, PER.C6® cells, and the like.[0003]For human application every pharmaceutical substance has to meet distinct criteria. To ensure the safety of biopharmaceutical agents to humans, for example, nucleic acids, viruses, and host...

AI Technical Summary

Benefits of technology

[0024]The current invention reports a method for concentrating immunoglobulin solutions to a concentration of more than 100 mg / ml. It has been surprisingly found that with a method according to the invention this can be achieved with low aggregate formation and in short time.

[0040]It has now surprisingly been found that with a tangential flow filtration (TFF) method according to the current invention in which the transmembrane pressure and cross-flow are variable during the filtration process and are adapted depending on the actual concentration of the immunoglobulin in the solution to be concentrated a concentrated immunoglobulin solution with low aggregate formation can be obtained in a short time. That is, it has surprisingly been found that aggregate formation during tangential flow filtration is low if a TFF method according to the invention is applied, i.e. a method in which during the filtration process the transmembrane pressure is changed and adapted according to the actual concentration of the antibody solution. The method according to the invention is a variable method compared to constant methods as known from the art, i.e. to methods in which the transmembrane pressure is adopted prior to the filtration process and is held constant during the entire tangential flow filtration process.

[0048]By observing flux versus ascending protein concentration there is no significant difference in the curves for processes at different Δp. But for the 3 bar mode a lower end concentration due to a high inlet pressure was observed. Compared to a concentration mode performed under a lower constant cross-flow (CF; 90 ml / min.) and a lower mean Δp (about 0.9 bar) a higher Δp of 1.2-1.8 bar contributes to an improved flux performance over time and a higher end concentration (TMP always 0.6 bar).

[0050]A TFF method has been developed with comparable overall process time compared to the stressing high inlet pressure mode (Δp=3 bar), based on TMP / CF-scouting experiments (see e.g. Luo, R., et al., Bioprocess Int. 4 (2006) 44-54). The method according to the invention has been developed to improve flux performance over time with reduced immunoglobulin aggregate formation, i.e. to combine a low aggregate formation and a short overall concentration time. During the developing of the method according to the invention a TMP and CF scouting was performed depending on the prevailing immunoglobulin concentration in the immunoglobulin solution to be concentrated. An method with adapted TMP and CF depending on the best flux profile at a given concentration was found. Without the disadvantage of high inlet pressure at the final stage of the concentration (see e.g. Dosmar, M., et al., Bioprocess Int. 3 (2005) 40-50) the method according to the invention showed a low aggregation burden in turbidity, LO, and DLS data for produced concentrates (see FIGS. 3 and 4). In addition a higher end concentration was achieved with the method according to the invention.

[0051]In the method according to the invention the transmembrane pressure and cross-flow are varied with respect to the actual concentration of the concentrated immunoglobulin solution. In one embodiment the method according to the invention is a variable tangential flow filtration method wherein the actual concentration of the immunoglobulin in the solution to be concentrated determines the applied transmembrane pressure and cross-flow. Thus, the transmembrane pressure and cross-flow are adjusted depending on the actual concentration of the immunoglobulin in order to reduce the stress applied and, thus, to reduce the formation of aggregated immunoglobulin molecules and to provide a short overall concentration time.

Problems solved by technology

Due to their chemical and physical properties, such as molecular weight and domain architecture including secondary modifications, the downstream processing of immunoglobulins is very complicated.

Furthermore short concentration times are favored to ensure smooth processes and short operating times. In this context imperfect TFF processes especially after final purification steps can cause sustained damage even affecting drug product.