Incorporation of automated buffer exchange empowers high-throughput protein and plasmid purification for downstream uses
The continued acceleration of time-to-market product development and rising demand for biotherapeutics have hastened the need for higher throughput within the biopharmaceutical industry. Automated liquid handlers (ALH) are increasingly popular due to flexible programming that enables processing of m...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2023-08-01
|
| Series: | SLAS Technology |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2472630323000055 |
| _version_ | 1797749101187563520 |
|---|---|
| author | Patrick A. Kates Jordan N. Cook Ryan Ghan Huey J. Nguyen Pongkwan Sitasuwan L. Andrew Lee |
| author_facet | Patrick A. Kates Jordan N. Cook Ryan Ghan Huey J. Nguyen Pongkwan Sitasuwan L. Andrew Lee |
| author_sort | Patrick A. Kates |
| collection | DOAJ |
| description | The continued acceleration of time-to-market product development and rising demand for biotherapeutics have hastened the need for higher throughput within the biopharmaceutical industry. Automated liquid handlers (ALH) are increasingly popular due to flexible programming that enables processing of multiple samples with an array of functions. This flexibility is useful in streamlining research that requires chromatographic procedures to achieve product purity for downstream analysis. However, purification of biologics often requires additional off-deck buffer exchange steps due to undesirable elution conditions such as high acid or high salt content. Expanding the capability of ALHs to perform purification in sequence with buffer exchange would, therefore, increase workflow efficiency by eliminating the need for manual intervention, thus expediting sample preparation. Here we demonstrate two different automated purifications using pipet-based dispersive solid-phase extraction (dSPE). The first is an affinity purification of His-tagged proteins from bacterial lysate. The second is an anion-exchange purification of plasmid DNA. Both methods are followed by buffer exchange performed by an ALH. Percent recoveries for the three purified recombinant proteins ranged from 51 ± 1.2 to 86 ± 10%. The yields were inversely correlated to starting sample load and protein molecular weight. Yields for plasmid purification ranged between 11.4 ± 0.8 and 13.7 ± 0.9 µg, with the largest plasmid providing the highest yield. Both programs were rapid, with protein purification taking <80 min and plasmid purification <60 min. Our results demonstrate that high-quality, ready-to-use biologics can be obtained rapidly from a crude sample after two separate chromatographic processes without manual intervention. |
| first_indexed | 2024-03-12T16:15:26Z |
| format | Article |
| id | doaj.art-4d87df2aaeae4e73b5d56a55af194fef |
| institution | Directory Open Access Journal |
| issn | 2472-6303 |
| language | English |
| last_indexed | 2024-03-12T16:15:26Z |
| publishDate | 2023-08-01 |
| publisher | Elsevier |
| record_format | Article |
| series | SLAS Technology |
| spelling | doaj.art-4d87df2aaeae4e73b5d56a55af194fef2023-08-09T04:33:28ZengElsevierSLAS Technology2472-63032023-08-01284243250Incorporation of automated buffer exchange empowers high-throughput protein and plasmid purification for downstream usesPatrick A. Kates0Jordan N. Cook1Ryan Ghan2Huey J. Nguyen3Pongkwan Sitasuwan4L. Andrew Lee5Integrated Micro-Chromatography Systems, Inc., 110 Centrum Drive, Irmo, SC 29063, United StatesIntegrated Micro-Chromatography Systems, Inc., 110 Centrum Drive, Irmo, SC 29063, United StatesHamilton Company, Inc., Reno, NV, United StatesIntegrated Micro-Chromatography Systems, Inc., 110 Centrum Drive, Irmo, SC 29063, United StatesIntegrated Micro-Chromatography Systems, Inc., 110 Centrum Drive, Irmo, SC 29063, United StatesIntegrated Micro-Chromatography Systems, Inc., 110 Centrum Drive, Irmo, SC 29063, United States; Corresponding author.The continued acceleration of time-to-market product development and rising demand for biotherapeutics have hastened the need for higher throughput within the biopharmaceutical industry. Automated liquid handlers (ALH) are increasingly popular due to flexible programming that enables processing of multiple samples with an array of functions. This flexibility is useful in streamlining research that requires chromatographic procedures to achieve product purity for downstream analysis. However, purification of biologics often requires additional off-deck buffer exchange steps due to undesirable elution conditions such as high acid or high salt content. Expanding the capability of ALHs to perform purification in sequence with buffer exchange would, therefore, increase workflow efficiency by eliminating the need for manual intervention, thus expediting sample preparation. Here we demonstrate two different automated purifications using pipet-based dispersive solid-phase extraction (dSPE). The first is an affinity purification of His-tagged proteins from bacterial lysate. The second is an anion-exchange purification of plasmid DNA. Both methods are followed by buffer exchange performed by an ALH. Percent recoveries for the three purified recombinant proteins ranged from 51 ± 1.2 to 86 ± 10%. The yields were inversely correlated to starting sample load and protein molecular weight. Yields for plasmid purification ranged between 11.4 ± 0.8 and 13.7 ± 0.9 µg, with the largest plasmid providing the highest yield. Both programs were rapid, with protein purification taking <80 min and plasmid purification <60 min. Our results demonstrate that high-quality, ready-to-use biologics can be obtained rapidly from a crude sample after two separate chromatographic processes without manual intervention.http://www.sciencedirect.com/science/article/pii/S2472630323000055 |
| spellingShingle | Patrick A. Kates Jordan N. Cook Ryan Ghan Huey J. Nguyen Pongkwan Sitasuwan L. Andrew Lee Incorporation of automated buffer exchange empowers high-throughput protein and plasmid purification for downstream uses SLAS Technology |
| title | Incorporation of automated buffer exchange empowers high-throughput protein and plasmid purification for downstream uses |
| title_full | Incorporation of automated buffer exchange empowers high-throughput protein and plasmid purification for downstream uses |
| title_fullStr | Incorporation of automated buffer exchange empowers high-throughput protein and plasmid purification for downstream uses |
| title_full_unstemmed | Incorporation of automated buffer exchange empowers high-throughput protein and plasmid purification for downstream uses |
| title_short | Incorporation of automated buffer exchange empowers high-throughput protein and plasmid purification for downstream uses |
| title_sort | incorporation of automated buffer exchange empowers high throughput protein and plasmid purification for downstream uses |
| url | http://www.sciencedirect.com/science/article/pii/S2472630323000055 |
| work_keys_str_mv | AT patrickakates incorporationofautomatedbufferexchangeempowershighthroughputproteinandplasmidpurificationfordownstreamuses AT jordanncook incorporationofautomatedbufferexchangeempowershighthroughputproteinandplasmidpurificationfordownstreamuses AT ryanghan incorporationofautomatedbufferexchangeempowershighthroughputproteinandplasmidpurificationfordownstreamuses AT hueyjnguyen incorporationofautomatedbufferexchangeempowershighthroughputproteinandplasmidpurificationfordownstreamuses AT pongkwansitasuwan incorporationofautomatedbufferexchangeempowershighthroughputproteinandplasmidpurificationfordownstreamuses AT landrewlee incorporationofautomatedbufferexchangeempowershighthroughputproteinandplasmidpurificationfordownstreamuses |