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Methods of producing polypeptides using a cell line resistant to apoptosis

A cell line and cell technology, applied in the field of cell line and cell culture, can solve the problem of few HEK293 cells found

Pending Publication Date: 2021-11-09
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Despite numerous reports describing CHO large-scale bioreactor culture and transfection, little has been found on HEK293 cells, and there is currently no long-term cultivation of HEK293 seed cultures in bioreactors to support routine high-throughput transfection. Reports that generate a large number of proteins

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  • Methods of producing polypeptides using a cell line resistant to apoptosis
  • Methods of producing polypeptides using a cell line resistant to apoptosis
  • Methods of producing polypeptides using a cell line resistant to apoptosis

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[0190] The present invention will be more fully understood by reference to the following examples. However, these examples should not be considered as limiting the scope of the invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes therefrom are suggested to those skilled in the art and are to be included within the spirit and purview of this application and the accompanying within the scope of the patent application.

example 1

[0191] Example 1: Generation and testing of a more stable HEK293 cell line

[0192]As described above, HEK293 cells are sensitive to shear stress and tend to develop low viability when cultured in bioreactors. It is assumed that cell lines resistant to apoptosis will exhibit higher productivity and more stable performance in bioreactors. An antiapoptotic HEK293 cell line (HEK293 DKO) was engineered to delete the pro-apoptotic genes Bax and Bak by using zinc finger nuclease technology (Cost et al., (2010) Biotechnol. Bioeng. 105:330-340). During apoptosis, Bax and Bak permeate the mitochondrial membrane, eventually causing activation of the caspase protein, which triggers programmed cell death (Taylor et al., (2008) Nat. Rev. Mol. Cell Biol. 9:231-241) . Deletion of Bax and Bak in CHO cell lines has previously been shown to correlate with higher culture survival and transfection titers (Macaraeg et al., (2013) Biotechnol. Prog. 29:1050-1058). Suppression or deletion of Bax a...

example 2

[0224] Example 2: Scale-up of HEK293 seed cultures

[0225] To efficiently generate cell populations to support large-scale (10L) transfection, HEK293 DKO seed cultures were grown in 35L controlled bioreactors rather than multiple shake flasks. A regularly subcultured (i.e., split every 3 to 4 days) seed culture bioreactor with a working volume of 20 to 35 L will provide sufficient 6 40 to 70 L of transfected cells were seeded per cell / mL. This enables high-throughput, large-scale transient production runs to be performed on a regular basis.

[0226] Different pH and dissolved oxygen (DO) controlled conditions were first evaluated in two 2L bioreactors before testing HEK293 DKO seed cultures in 35L bioreactors. Bioreactor #1 used a pH set point of 7.0 with an insensitivity zone ±0.03 and a DO set point of 30%; these are typical conditions for CHO stable cell line cultures (Li et al., (2010) mAbs.2:466- 477; Li et al., (2012) Biotechnol. Bioeng. 109:1173-1186; Yuk et al., (2...

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Abstract

Provided herein are cell lines (e.g., HEK293 cell lines) that comprise a loss-of-function mutation in each of the human Bax and Bak genes, as well as cell cultures comprising the cell lines. The cell lines and / or cell cultures may find use, e.g., in methods for producing a recombinant polypeptide (such as an antibody or antigen-binding fragment thereof) or a viral vector.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 784,051, filed December 21, 2018, which is hereby incorporated by reference in its entirety. technical field [0003] The present invention relates to methods for producing recombinant polypeptides or viral vectors, and cell lines and cell cultures that can be used, for example, in said methods. Background technique [0004] Monoclonal antibodies (mAbs) and other recombinant proteins have been established as successful therapeutics for many disease indications, including immunology, oncology, neuroscience, and others (see, e.g., Reichert (2017) mAbs. 9:167-181; Singh et al. (2017) Curr. Clin. Pharmacol. 13:85-99). With more than 300 mAbs in development in the biotechnology industry, the mAb market is expected to expand to 70 mAb products by 2020 (Ecker et al., (2015) mAbs. 7:9-14). As the industry expands and targets become more co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/67C07K14/47C12N5/071
CPCC12N15/67C07K14/4747C12P21/02C12N2511/00C12N15/85C12N5/0686C12N15/86C12N2510/02
Inventor T·A·阿瑞纳A·W·黄
Owner F HOFFMANN LA ROCHE & CO AG
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