Optimized dual assay for the transgenes selection and screening in CHO cell line development for recombinant protein production
OBJECTIVE: To develop a simple robust methodology of screening multiple CHO cell clones secreting recombinant proteins to assess their specific productivity.
RESULTS: We developed a dual assay based on immunoassay measurements of a recombinant protein expression combined with staining of viable cells with resazurin. Following this approach, colonies can be simultaneously assessed for cell growth rate and for production of a recombinant protein. Combination of these two assays enables to estimate productivity of a recombinant protein per cell from the very early stages of a cell line development process (CLD) and exclude poor producers from further steps. Comparison of the dual assay with a standard CLD protocol followed by only analysis of protein expression level showed at least 10-20% increase in the amount of clones that can be included into pool of high-producers at early stages. This shortens duration of a typical CLD scheme from 23 to 19 weeks.
CONCLUSIONS: Our method: (i) allows to include into workflow clones that demonstrate slow growth during single cell cloning but producing high amounts of a target protein, which otherwise would be lost in standard protocols of cells screening; (ii) can be applied for testing of DNA vectors for transfection and protein production; (iii) can be used for monitoring the heterogeneity of cell population and analysis of stable pools productivity.
| Medienart: |
E-Artikel |
|---|
| Erscheinungsjahr: |
2019 |
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| Erschienen: |
2019 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:41 |
|---|---|
| Enthalten in: |
Biotechnology letters - 41(2019), 8-9 vom: 18. Sept., Seite 929-939 |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Beketova, Elena V [VerfasserIn] |
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| Links: |
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| Themen: |
CHO DG44 |
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| Anmerkungen: |
Date Completed 22.11.2019 Date Revised 22.11.2019 published: Print-Electronic Citation Status MEDLINE |
|---|
| doi: |
10.1007/s10529-019-02711-4 |
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| funding: |
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|---|---|
| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM299340651 |
|---|
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| 100 | 1 | |a Beketova, Elena V |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Optimized dual assay for the transgenes selection and screening in CHO cell line development for recombinant protein production |
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| 500 | |a Date Completed 22.11.2019 | ||
| 500 | |a Date Revised 22.11.2019 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a OBJECTIVE: To develop a simple robust methodology of screening multiple CHO cell clones secreting recombinant proteins to assess their specific productivity | ||
| 520 | |a RESULTS: We developed a dual assay based on immunoassay measurements of a recombinant protein expression combined with staining of viable cells with resazurin. Following this approach, colonies can be simultaneously assessed for cell growth rate and for production of a recombinant protein. Combination of these two assays enables to estimate productivity of a recombinant protein per cell from the very early stages of a cell line development process (CLD) and exclude poor producers from further steps. Comparison of the dual assay with a standard CLD protocol followed by only analysis of protein expression level showed at least 10-20% increase in the amount of clones that can be included into pool of high-producers at early stages. This shortens duration of a typical CLD scheme from 23 to 19 weeks | ||
| 520 | |a CONCLUSIONS: Our method: (i) allows to include into workflow clones that demonstrate slow growth during single cell cloning but producing high amounts of a target protein, which otherwise would be lost in standard protocols of cells screening; (ii) can be applied for testing of DNA vectors for transfection and protein production; (iii) can be used for monitoring the heterogeneity of cell population and analysis of stable pools productivity | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a CHO DG44 | |
| 650 | 4 | |a DHFR | |
| 650 | 4 | |a Optimization | |
| 650 | 4 | |a Recombinant mAbs | |
| 650 | 4 | |a Recombinant proteins | |
| 650 | 4 | |a Resazurin | |
| 650 | 4 | |a Selection | |
| 650 | 7 | |a Recombinant Proteins |2 NLM | |
| 700 | 1 | |a Ibneeva, Liliia R |e verfasserin |4 aut | |
| 700 | 1 | |a Abdulina, Yulia A |e verfasserin |4 aut | |
| 700 | 1 | |a Dergousova, Elena A |e verfasserin |4 aut | |
| 700 | 1 | |a Filatov, Vladimir L |e verfasserin |4 aut | |
| 700 | 1 | |a Kozlovsky, Stanislav V |e verfasserin |4 aut | |
| 700 | 1 | |a Shilov, Evgeny S |e verfasserin |4 aut | |
| 700 | 1 | |a Datskevich, Petr N |e verfasserin |4 aut | |
| 700 | 1 | |a Rozov, Fedor N |e verfasserin |4 aut | |
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