Cathepsin D Knockout Mammalian Cells for Antibody Peptide Conjugate Production
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Solution Overview
Problem
Host cell proteins, particularly proteases like cathepsin D, clip peptides during recombinant antibody production, leading to damaged peptides and loss of efficacy, making it difficult to remove process-related impurities.
Innovation Solution
Engineering mammalian cell lines with reduced or eliminated cathepsin D expression through genetic modification, such as CRISPR or zinc-finger technology, to produce antibodies with low contaminating protease levels, followed by purification and peptide conjugation at specific sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If host cell lines are used for antibody production, then antibody production is achieved, but protease contamination occurs leading to peptide clipping and loss of efficacy
Solution Approach 1:
The patent extracts and removes the harmful cathepsin D protease from the host cell line through genetic engineering. Specifically, the cell line is modified to have reduced or eliminated cathepsin D expression, thereby removing the source of peptide clipping while maintaining antibody production capability.
Solution Approach 2:
The patent changes the biochemical parameter of protease expression in the host cell line by using cathepsin D knock-out or knock-down cell lines. This parameter change reduces protease contamination from detectable levels to below detection limits, preventing peptide clipping during antibody production.
2Productivity
If conventional host cell lines are used, then antibody production is achieved, but process-related impurities are difficult to remove
Solution Approach 1:
The patent extracts the source of process-related impurities by removing cathepsin D expression from the host cell line. This eliminates the primary source of proteolytic impurities that would otherwise require complex purification steps to remove.
Solution Approach 2:
The patent applies preliminary action by genetically modifying the host cell line before antibody production to prevent impurity formation at the source. By using cathepsin D knock-out cell lines from the outset, the patent prevents rather than addresses impurity removal, simplifying downstream processing.
3Productivity
If peptide conjugation is performed with conventional antibodies, then conjugate production is achieved, but peptide clipping occurs reducing conjugate quality
Solution Approach 1:
The patent applies preliminary anti-action by pre-modifying the host cell line to eliminate cathepsin D expression before peptide conjugation occurs. This preventive measure stops peptide clipping before it can happen during the conjugation process, ensuring high conjugate quality.
Solution Approach 2:
The patent changes the proteolytic activity parameter of the host cell line to zero or near-zero by using cathepsin D knock-out cell lines. This parameter change ensures that peptides remain intact during antibody production and subsequent conjugation, maintaining manufacturing precision.
Data Source
AI summary
The present invention relates to mammalian cells modified to provide for reduced cleavage of peptides that will be conjugated to polypeptides expressed by the cells. In particular, the invention relates to a method of producing an antibody peptide conjugate, the method comprising: a) expressing the antibody in a mammalian cell wherein the mammalian cell is a cathepsin D knock out cell, and wherein the antibody comprises a cysteine or non-canonical amino acid amino acid substitution at one or more conjugation site(s); b) purifying the antibody; and c) conjugating a peptide to the antibody at the conjugation site(s).