Early post-transfection isolation of cells (EPIC) for biologics production

a cell and post-transfection technology, applied in the field of early post-transfection isolation of cells (epic) for biologics production, can solve the problems of drug-based selection time-consuming, negative impact on clonal stability, and inability to meet the needs of targeted populations, and achieve the effect of improving the production of target polypeptides

Inactive Publication Date: 2018-05-03
GENZYME CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013]In some embodiments of any one of the methods provided, at least one of the clonal populations of the one or more single transfected host cells yields...

Problems solved by technology

Selection agent-based methods may affect the viability or growth rate of selected populations or may have a negative impact on clonal stability.
Such drug-based sel...

Method used

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  • Early post-transfection isolation of cells (EPIC) for biologics production
  • Early post-transfection isolation of cells (EPIC) for biologics production
  • Early post-transfection isolation of cells (EPIC) for biologics production

Examples

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example 1

Sorting for Early Post-Transfection Isolation of Cells (EPIC)—Proof of Concept

[0143]This example demonstrates the feasibility of a method of sorting to target an unselected transfected early-expressing population for bulk enrichment prior to selection. This method of sorting is called “short sorting” or “EPIC” (Early Post-transfection Isolation of Cells) and is designed to sort-isolate, or bulk enrich, early reporter expression shortly after transfection. EPIC may significantly reduce selection timelines and / or improve productivity of the resulting heterogeneous population. Experiments have been performed to investigate the reporter expression profile of a transfected population throughout the course of a nucleotide-deficient selection process. FIG. 1A depicts a general scheme for EPIC. FIG. 1B shows the early expression of the reporter gene during the nucleotide-deficient selection process. These offset histograms demonstrate that early expression (e.g. day 3-4) is positive and sor...

example 2

Sorting for Early Post-Transfection Isolation of Cells (EPIC)—Producer Cell Pool Generation

[0159]EPIC was initially attempted using mAb#1 in which CHO cells were transfected and given 2 days to recover, after which 0 nM MTX selection was initiated to establish early expression. Four days after transfection, early expression of CD52 cell surface reporter was targeted for sort isolation (EPIC). Sorting targeted only positive expression which was collected as a bulk enriched population of about 1 million cells which was then allowed to continue selection in nucleotide-deficient media (0 nM MTX). As a control, a non-sorted transfection was allowed to continue selection via standard selection procedure. As shown in FIG. 4, by day 8 sorting for EPIC yielded a slightly enriched population as seen by CD52 reporter expression as compared to standard selection. As selection of both populations continued, however, this small EPIC sub-population became more prominent over time. In fact, the CD5...

example 3

Sorting for Early Post-Transfection Isolation of Cells (EPIC)—Clone Generation

[0162]The EPIC-generated pool of Example 2 was next used to generate clones using FLARE as previously described (see, e.g., Cairns, V. et al. (2011) Utilization of Non-AUG Initiation Codons in a Flow Cytometric Method for Efficient Selection of Recombinant Cell Lines, Biotechnol Bioeng 108(11):2611-2622). Briefly, FLARE was used to isolate and single cell plate the top 3-5% of reporter-expressing cells from each pool using FACS. Expanded clones were then screened (taking top 30% positive expressers), again using FLARE, to identify only the top tier clones to expand for target polypeptide titer evaluation. As shown in FIG. 6, top expressing EPIC-generated clones achieved similar titers to those of best clones from traditional methods, e.g., using MTX-amplified pools (near 2.0 g / L). Results demonstrated that using EPIC to isolate early expression populations prior to selection is a viable alternative to trad...

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Abstract

Provided herein are methods for selecting a population of cells expressing a target polypeptide. In some aspects, the disclosure provides methods for sorting and selecting populations of transfected host cells based on their early expression of a selectable polypeptide. In certain embodiments, the sorting is performed using fluorescence-activated cell sorting or magnetic-activated cell sorting based on the selectable polypeptide. Such selection methods can be further utilized to generate clonal populations of producer cells, e.g. for large-scale manufacturing of a target polypeptide of interest.

Description

RELATED APPLICATION[0001]This application claims benefit of United States Provisional Patent Application No. 62 / 405,392, filed on Oct. 7, 2016, the entire content of which is incorporated herein by reference.SEQUENCE LISTING[0002]The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 19, 2017, is named 594097_SA9_179C_ST25.txt and is 15,429 bytes in size.BACKGROUND[0003]Methods for selection of producer cell populations and cell clones are imperative for the manufacturing of biologics, such as antibodies and fusion proteins. Such methods generally rely on use of a selection agent, such as methotrexate (MTX) or methionine sulphoximine (MSX), to bias and amplify the production of biologics. Selection agent-based methods may affect the viability or growth rate of selected populations or may have a negative impact on clonal stability. Such drug-b...

Claims

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Application Information

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IPC IPC(8): C12P21/00C12N15/85C07K14/705C07K14/435
CPCC12P21/00C12N15/85C07K14/70592C07K14/435C07K14/70596C07K14/43504C12N5/0087C12N5/10G01N15/14G01N2015/149
Inventor CAIRNS, VICTOR R.DEMARIA, CHRISTINEVITKO, JASON
Owner GENZYME CORP
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