| 摘要 |
The present invention is directed to methods for the separation of a molecule of interest from a solution containing the molecule using dual stage tangential-flow ultrafiltration (“TFF”). In particular, the methods of the invention are directed to the processing of crude feed streams such as conditioned cell culture supernatant to dramatically reduce contaminant and/or impurity levels prior to subsequent, i.e., downstream, refining unit operations. The methods of the invention may be used in the processing of a crude feed stream from biological production systems such as fermentation or other cell culture process, and may further eliminate the need for time consuming impurity precipitation (e.g., pH driven) and/or precipitate filtration processes prior to downstream processes that are sensitive to high impurity loads such as chromatographic unit operations. The disclosed dual stage TFF process combines at least two TFF unit operations that may be advantageously conducted at a pH that corresponds to or is about that of the pH of the feed stream, e.g., a cell culture supernatant, typically a pH of 7.5±1.0. The use of the TFF unit operations to supplement, improve or replace traditional processes for purification of proteins of interest for a feed stream may represent significant savings in both direct and indirect processing costs, For example, in addition to indirect savings by eliminating precipitation and precipitate filtration processes, the reduction in impurity loads effected by the dual stage TFF unit operations may result in indirect savings by improving downstream column performance, e.g., chromatographic separation, dynamic binding capacity, operational lifetime and/or a reduction of the required column size. In particular embodiments, the methods of the invention are used in processes for the purification of immunoglobulin molecules, e.g., antibodies, which processes are devoid of affinity purification steps, e.g., protein A affinity chromatography purification. |
