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Cell strain for producing biosimilar drug of Ustekinumab and production method thereof

A cell line and ovarian cell technology, applied in the field of biosimilar drugs for the production of ustekinumab, can solve the problems of physical and chemical properties, differences in biological activity, metabolic behavior in vivo, inability to pass similarity, low similarity, etc.

Active Publication Date: 2021-05-28
QYUNS THERAPEUTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But in general, the same monoclonal antibody expressed by different types of host cells, even if the amino acid sequence is the same, may have structural differences due to different glycosylation methods, etc. The differences between biosimilars and the original drugs lead to low similarity and cannot pass the similarity evaluation
This is a major difficulty in the development of biosimilars, and it is also an important reason why there is little information about its biosimilars, although the patent protection of ustekinumab is about to expire

Method used

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  • Cell strain for producing biosimilar drug of Ustekinumab and production method thereof
  • Cell strain for producing biosimilar drug of Ustekinumab and production method thereof
  • Cell strain for producing biosimilar drug of Ustekinumab and production method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: vector construction

[0049] The heavy chain amino acid sequence (SEQ ID NO: 1) and light chain amino acid sequence (SEQ ID NO: 3) of ustekinumab were determined through public information query and peptide analysis. (Such as figure 1 )

[0050] The heavy chain amino acid sequence and light chain amino acid sequence of QX001S, which is a biosimilar drug of ustekinumab, are completely identical to ustekinumab.

[0051] Using the heavy and light chain amino acid sequence of ustekinumab as a template, the heavy and light chain gene sequence of QX001S was designed, and the expression plasmid was constructed.

[0052] The heavy chain gene sequence of the codon-optimized QX001S is SEQ ID NO: 2 (as shown in Figure 4).

[0053] The light chain gene sequence of the codon-optimized QX001S is SEQ ID NO: 4 (as shown in Figure 5).

[0054]In the expression plasmid system, the codon-optimized heavy chain gene and light chain gene of QX001S were respectively construc...

Embodiment 2

[0056] Embodiment 2: Screening of cell lines

[0057] Confirm that the constructed QX001S expression vector gene sequence and expressed protein are correct, and use CHO-S as the host cell to construct the stable expression cell line of QX001S. CHO-S host cells were purchased from ThermoFisher (formerly Invitrogen, Cat. No. A13696-01), recovered and cultured CHO-S TM cell. After the linearized expression vector is electroporated into the host cell, it is randomly integrated into the genome of the host cell through the homology arm on the vector.

[0058] Using glutamine synthetase as a screening marker, a CHO-S cell line with stable, high-yield expression and physicochemical properties consistent with ustekinumab was obtained by pressurized screening with methionine sulfone methylene (MSX).

[0059] 1. Through step-by-step amplification, ELISA detection and other operations, for the cell clones cultured in 40 plates and 96-well plates, a high-yielding cell pool (mixed cell cl...

Embodiment 3

[0065] Embodiment 3: Pharmaceutical similarity research

[0066] The pharmaceutical similarity between QX001S expressed by the CHO-S cell line of the above clone 4 and ustekinumab was studied. We comprehensively compared the primary structure, secondary and advanced structure, translation of QX001S and ustekinumab The similarities and differences in post-modification, charge heterogeneity, aggregates and degradation products, in vitro biological activity, and accelerated stability show that the two are highly similar in pharmaceutical research. The QX001S expressed by the CHO-S cell line is basically consistent with ustekinumab in 71 of the 78 indicators listed in Table 1 below.

[0067] The indicators that have differences between the two are mainly in post-translational modification (there are differences in 5 indicators), especially as determined, the proportion of sialylated components of ustekinumab is between 11.87% and 25.56%, while the CHO -S cell line expresses QX001...

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Abstract

The invention discloses a cell strain for producing a biosimilar drug of Ustekinumab and a production method thereof. The Chinese hamster ovary cell S cell strain is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: C2020232. The cell strain expresses a fully human monoclonal antibody resisting a p40 sub-unit shared by human IL-12 and human IL-23. The fully humanized monoclonal antibody for resisting the p40 sub-unit shared by the human IL-12 and the human IL-23 is a biosimilar drug of Ustekinumab, not only shows high consistency with the Ustekinumab in pre-clinical research, but also passes pharmacokinetic bio-equivalence and safety similarity evaluation in clinical research, and the provided biosimilar drug of Ustekinumab is the first one that enter the clinical test period in China, is the only one that completes the I stage clinical test, and is one of the biosimilar drugs of Ustekinumab, which are the fastest in application in the international.

Description

technical field [0001] The invention relates to the field of antibody medicine. Specifically, the present invention relates to a drug that can be used for the production of Ustekinumab (Ustekinumab, trade name ) Chinese hamster ovary cell S (CHO-S) cell line of a biosimilar drug and a method for producing a biosimilar drug of ustekinumab using the CHO-S cell line. Background technique [0002] Ustekinumab (ustekinumab, trade name ) developed by Johnson & Johnson, is a fully human monoclonal antibody targeting the p40 subunit shared by human IL-12 and IL-23. IL-12 and IL-23 are heterodimeric cytokines expressed by activated antigen-presenting cells, and IL-12 can activate NK cells to promote CD4 + T cells differentiate into helper T cell 1 (Th1), and IL-23 can activate the helper T cell 17 (Th17) pathway. Dysregulation of IL-12 and IL-23 is closely related to immune-related diseases. Ustekinumab inhibits IL-12 and IL-23-mediated signaling and cytokines by blocking the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C07K16/24
CPCC12N5/0682C12N15/85C07K16/244C12N2510/02C07K2317/21Y02A50/30A61K2039/505C12N2510/04C07K2317/31
Inventor 徐正学李涛陈寅黄文俊王轶乔怀耀房敏吴亦亮张梦丹
Owner QYUNS THERAPEUTICS CO LTD
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