Optimizing Expression Systems - Industry experts from Fujifilm Diosynth and Boehringer Ingelheim discuss methods for optimizing protein expression in bacterial and mammalian cell lines. - BioPharm


Optimizing Expression Systems
Industry experts from Fujifilm Diosynth and Boehringer Ingelheim discuss methods for optimizing protein expression in bacterial and mammalian cell lines.

BioPharm International
Volume 25, Issue 7, pp. 24-27


Anne B. Tolstrup, Barbara Enenkel, Stefan Schlatter, Jochen Schaub, Harald Bradl, Anja Puklowski, Patrick Schulz, Anurag Khetan and Hitto Kaufmann, Mammalian Cell Line and Process Development in Process Science, Boehringer Ingelheim

Figure 1A: Elements of Boehringer Ingelheirm’s BI-Hex expression system. (FIGURES 1A–3A COURTESY OF BOEHRINGER INGELHEIM)
Manufacturing of new biological entities (NBEs) such as monoclonal antibodies (mAbs) and bispecific derivatives typically depends on mammalian expression because of the necessary post-translational modification of these types of molecules. To meet today´s demands of short and cost-efficient development, the expression system employed should be robust, high-titered, and fast. The BI-HEX mammalian expression platform developed by Boehringer Ingelheim meets these requirements, and is comprised of four interlinked elements (see Figure 1):
  • A proprietary vector system
  • A Chinese hamster ovary (CHO) host cell line lacking the dhfr gene
  • A proprietary medium and
  • A complete upstream and downstream manufacturing process.

Behind each element, there is a panel of choices that can be exploited for further optimization of the individual NBE. For example, a panel of BI-HEX expression vectors are at hand comprising different IgG isotypes, different genetic elements, and different frameworks. Choice of the right vector constellation is important for optimization of the desired effector function as well as for the manufacturability of the NBE.

Figure 2A: Relative distributions of different glycoforms produced in HEX1 and HEX2 Chinese hamster ovary (CHO) cell lines.
The glycosylation structures on the mAb influence the effector function of the molecule. For example, antibody-dependent cell cytotoxicity is enhanced if the fucose content of the N-linked carbohydrate structure attached to Asn297 in the constant region of the molecule is reduced. The BI-HEX platform comprises two substrains of the host cell line, HEX1 and HEX2. Both cell lines are derivatives of the CHO DG44 cell line originally established in 1980, but they differ in their glycoprofiles (1). HEX1 has a higher content of A2FG0 and also a higher level of defucosylated carbohydrates compared with HEX2 which has a higher A2FG1 content (see Figure 2A). This allows preselection of a more optimal glycoprofile dependent on the desired function of a given NBE. Furthermore, for development of biosimilars, where it is critically important to match the originator molecule with respect to all clinically relevant product quality attributes, the platform provides the option to preselect a profile being most similar to the originator molecule.

Figure 3A: Comparison of titers resulting from different feed strategies.
Having generated the stable BI-HEX production cell line based on the optimal vector constellation and host cell substrain, the next important element is upstream process development. Based on this platform process, a fast and robust design-of-experiments-driven approach for media and process development can be used. This part of the BI-HEX expression platform involves the ability to manipulate or influence the product quality, such as the fine-tuned level of antibody-dependent cell-mediated cytotoxicity (ADCC), is highest—provided that the right vector and host cell line were selected from the start. Bioreactor parameters such as pH settings, stirring, and gassing as well as media adjustments, composition, and timing of the feed are all elements that have a high impact on product quality as well as on product titers. A significant increase in titer is typically seen during this part of the optimization and high titers of 5–8 g/L have been reached with BI-HEX (see Figure 3A).

Figure 4A: Comparison of antibody-dependent cell-mediated cytotoxicity by antibodies produced in an unmodified BI Hex expression system, and antibodies produced using GlyMaxX technology.
A recent addition to the BI-HEX platform is the generation of a BI-HEX cell line modified to produce antibodies with very low levels of fucose. This is achieved by means of the GlymaxX technology developed by ProBioGen with whom BI entered into a collaboration agreement in 2011. The BI-HEX GlymaxX antibodies exhibit a significant increase in ADCC activity which is desirable, for example, in cancer indications (see Figure 4A).

Looking into the future, further automation of all parts of the BI-HEX platform, including miniaturization of clone and process screening as well as media and feed optimization is in the pipeline. Also, use of disposable systems wherever possible is being pursued to leverage the fast and flexible turnaround of such systems.


1. G. Urlaub and L.A. Chasin, Proc. Natl. Acad. Sci. USA 77 (7), 4216–20 (1980).

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