Tech Review

Bispecific antibodies & single-use devices

06.03.2012

The use of bispecific antibodies recognising two different targets may allow improvement of clinical efficacy. A bispecific antibody for two growth factors that stimulate the formation of blood vessels – Ang2 (Angiopoietin-2) and VEGF-A (Vascular Endothelial Growth Factor A) – has been developed by Roche Pharma Research and Early Development (pRED). For the first time, single-use bioreactors (SUBs) in Roche’s fermentation plant have been used for production of this bispecific antibody for toxicological studies, successfully combining two emerging technologies.

Bispecific antibodies incorporate the antigen­-binding properties of two different monospecific antibodies. The basic principle underlying this technology involves transformation of the host cell line with the genetic information encoding for four different protein chains. Although the cell line produces the desired combination, it may also yield nine other antibody combinations. In order to avoid the mispairing of heavy and light chains, the protein chains have to be altered in a way that favors the formation of the correct molecule. This is achieved by two pivotal changes: first, the CH3 domains of two heavy chains are modified so that one exhibits a knob and the other a hole. The second alteration ensures the correct pairing of the light chains to their corresponding heavy chains. This is done via the crosswise exchange of CH1 and C in one arm of the antibody to produce a CrossMAb.
The CrossMAb <Ang2/VEGF>
The bispecific antibody binding both Ang2 and VEGF-A is expected to weaken and ultimately eliminate a tumour by reducing the supply of nutrients from blood vessels, thereby suppressing tumour growth. Blood vessel formation requires a complex sequence of steps stimulated by Ang2 and VEGF-A. Because they are essential for angiogenesis, inactivating these two growth factors should allow the bi­specific antibody for Ang2 and VEGF-A to strike the tumour effectively.
Different challenges had to be addressed when developing the CrossMAb <Ang2/VEGF>. The most important prerequisite was that the four protein chains be expressed in the correct, stable stoichio­metric relationship. To do this, two DNA plasmids, each carrying the genetic information of one light and one heavy chain, were transferred into CHO cells. Both plasmids were mixed in different ratios, and the resulting cell clones were analysed. Twelve of the best candidates were subcloned and 23 of these subclones were characterized intensely in shake flasks and 2L bioreactors. The most important parameters for selecting the clones and subclones were IgG quantification and the ana­lysis of unwanted byproducts, such as incomplete antibodies or hetero­dimers of two light chains. The best clones were further tested for expression stability, product quality, glycosylation pattern, structural integrity, anti­gen binding and bi-functionality. In order to produce material for preclinical toxico­logical studies, the selected clone was cultivated in 100L and 250L SUBs. During these fermentation processes, cells reproducibly reached densities of up to 8 x 106 mL-1 and maximal product concentrations of 4g/L-1.
Design of the SUB plant
The conceptual design phase of the SUB plant revealed that cell growth and product yield in different types of SUBs are comparable to those in stainless steel fermenters. The rocker and stirred tank vessels that were ultimately chosen are combined in such a way that their split ratio is 1:5, and each pre-fermentation line supports two main fermenters. The plant consists of 50 L, 100 L and 250 L fermenter lines (see Figure). Because each reactor has ports for feeding nutrients, all of the pre-fermenters can also function as main fermenters. Flexible tubes connect the different reactors. Depending on the desired combination, split ratios between 1:2 and 1:10 can be realized.
Fermentation systems

and processes
The cell-culture media are prepared in stainless steel vessels with working volumes of 60-600L for single or multiple fermentations. Finally, the media are passed through sterile filters into storage bags or directly into fermenter bags. The cells are cultivated in 10-20L bags, which are fixed on a rocking system that is aerated through the head space. SUBs with a working volume of 50, 100 und 250L are cylindrical, and contain a marine impeller for mixing the culture. The bag is aerated via submerged gas injection. All fermentation units are mounted on wheels and are supplied with the required media (gases, electricity) from the ceiling, which allows them to move when desired.
Temperature, pH and pO2 are recorded and controlled in every fermentation bag. Some types of bags contain only optical sensors with a disposable sensor patch to measure pH and pO2, while others also allow users to insert electrochemical probes. Additional single-use sensors control pressure in the bags and enable monitoring of foam formation. The pressure control prevents overpressure, which can cause disruption of bags. Online and offline data are recorded and visualised, with different parameters of several fermenters shown in a single plot. This allows it to make fast comparisons, even between ongoing fermentations. 10-50L cultures are harvested using depth filtration or swing-head centrifuges with a capacity of 6-12L. In 100-250L fermentations, the cells are removed by separators.
The reactors support Roche Pharma projects by culturing many cell lines and applying different process techniques, allowing for batch and fed-batch processes with up to three nutrient feeds. To provide proteins for early project phases, mammalian cell lines are transfected with DNA plasmids, or insect cells are infected with baculoviruses. Murine hybridoma lines can also be cultivated in SUBs. During late project phases, stably-expressing CHO lines are used for producing material for preclinical toxicological studies, as is the case with the CrossMAb <Ang2/VEGF>. Bioprocesses with stable CHO lines are transferred to the GMP area, equipped with identical 250L SUBs and meet the demand for the early clinical phases. Implementing SUB technology at Roche’s site in Penzberg established a fermentation platform producing enough material to cover demand from early project phases up through clinical studies.
Outlook
The CrossMAb <Ang2/VEGF> proves that these new antibody formats can be manufactured for therapeutic applications. Further development of these formats will lead to proteins that are designed for specific indications and that clearly differ from naturally occurring antibodies. SUB technology allows it to shorten development timelines and to use identical processes for supply of GLP-tox and early clinical phases. Fermenters on a scale of about 10 m³ are needed for late clinical phases and market supply. Stainless steel reactors will continue to be used for these applications, as these dimensions are still difficult to achieve with SUBs.

References
Eisenkrätzer D, Herrmann A, Lipok G , Caudill U, Guilmaille O, Paul W, Traverse M , Reithmeier R, De Roo E (2010) Roche’s platform for early process development & transfer based on single use bioreactors. GVC/DECHEMA Tagung Bioprozessorientiertes Anlagendesign, 2010, Nürnberg.
Herrmann A, Eisenkrätzer D, Paul W, Lipok G, Kuske S (2011) Implementation of single-use technology into existing facilities. 4th European Biotechnology Day 2011, Neuss.
Klein C & Schaefer W (2009) Bivalent, bispecific anibodies. WO 2009080253 A1.
Kohnert (2011) Expression of bispecific antibodies in CHO Cells. IBC's 7th Annual Cell Line Development & Engineering, 2011, Boston.
Paul W, Hauser F, Schwald C, Reithmeier R, Herrmann A, Eisenkrätzer D (2011) Single-use sensors in the environment of disposable bioreactors with application in fermentation processes. BioTech 2011, Wädenswil.
Ridgway JB, Presta LG, Carter P (1996) “Knobs-into-holes” engineering of antibody CH3 domains for heavy chain hetreodimerisation. Protein Eng 9: 617-621.
Schaefer W, Regula JT, Bähner M, Schanzer J, Croasdale R, Dürr H, Gassner C, Georges G, Kettenberger H, Imhof-Jung S, Schwaiger M, Stubenrauch KG, Sustmann C, Thomas M, Scheuer W, Klein C (2011) Immunoglobulin domain crossover as a generic approach fort the production of bispecific IgG antibodies. Proc Natl Acad Sci USA 108(27): 11187-11191


Contact
Stefan Koch, Ingo Gorr und Wolfgang Paul
Roche Diagnostics GmbH, Biologics Research
Pharma Research and Early Development (pRED)
Stefan Koch@roche.com


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