CN-112980659-B - Systems, methods, and devices for automatically separating nucleic acids from proteins

Abstract

Purification of target biomolecules, such as nucleic acids or proteins, from biological sources is a time consuming process, which is usually performed by a skilled technician or scientist due to the highly technical nature of the work. The systems, devices, and methods disclosed herein enable automated biological treatment and purification of target biomolecules in biological sources. For example, the invention provides an instrument and disposable purification cartridge for automatically isolating and purifying nucleic acids (e.g., plasmid DNA from bacterial cultures) or separating proteins from any biological sample. Such exemplary instruments and purification columns can work cooperatively to release, mix and move the target biomolecule and various reagents and buffers in time during purification of the target biomolecule, resulting in less manual supervision of the purified target biomolecule than conventional methods.

Inventors

• A. Frasov
• W. Luciano
• J. Greck
• J. JONES
• D. Theron
• S. Gersh
• B. Vaida
• A. Basta
• A. EVANS
• DERBY KEVIN
• R. Setquist
• T. Bata
• A. Cox
• M. Meyernik
• J. Insley
• E. Gunter
• R. SMITH
• S. Tiller
• C. ADAMS
• K. Kruger
• R. SCHNEIDER

Assignees

• 生命科技股份有限公司

Dates

Publication Date

20260512

Application Date

20201217

Priority Date

20191218

Claims (20)

1. 1. A system comprising an apparatus for automatically purifying a target biomolecule from a biological sample, comprising: a purification column, the purification column comprising: An input reservoir for containing a biological sample; A first biological treatment assembly in fluid communication with the input reservoir and the lysis buffer reservoir, the first biological treatment assembly for producing a lysate comprising a target biomolecule; a second biological treatment assembly in fluid communication with the first biological treatment assembly and the first elution buffer reservoir, the second biological treatment assembly including a target biomolecule binding filter for retaining a target biomolecule. A container in fluid communication with the second biological processing assembly, the container for housing an output container containing a target biological molecule from the second biological processing assembly; a housing having an interior compartment sized and shaped to house the purification column; a pump assembly disposed within the interior compartment for providing a pumping action through peristaltic movement, and A clamping mechanism disposed within the interior compartment and configured to move between an open position in which the interior compartment is accessible and a closed position in which the clamping mechanism clamps onto an inserted purification column to enable processing of the biological sample, wherein the clamping mechanism operates to compress the purification column and thereby assist in fluid sealing the purification column.
2. 2. The system of claim 1, wherein the biological sample is a cell culture, a clinical sample, an environmental sample, or a food sample.
3. 3. The system of claim 1, further comprising an optical density detector window disposed between the input reservoir and the first biological processing assembly, through which an optical density of the biological sample is detectable.
4. 4. The system of claim 1, wherein the first biological treatment assembly comprises a clarification filter.
5. 5. The system of claim 4, wherein the clarification filter is in fluid communication with the input reservoir and the lysis buffer reservoir, the clarification filter for separating a target nucleic acid containing portion of the biological sample from a first waste portion of the biological sample.
6. 6. The system of claim 4, wherein the first biological treatment assembly comprises a cell capture filter or a concentrate filter, wherein the cell capture filter or concentrate filter is disposed upstream of the clarification filter.
7. 7. The system of claim 6, wherein the cell capture or concentration filter is in fluid communication with the input reservoir and the lysis buffer reservoir, the cell capture or concentration filter for separating a target nucleic acid-containing portion of the biological sample from a first waste portion of the biological sample.
8. 8. The system of claim 6, wherein the lysis buffer reservoir is in fluid communication with the cell capture filter, enabling back flushing of the cell capture filter and back flushing fluid to enter the clarification filter.
9. 9. The system of claim 8, further comprising a first mixing chamber disposed between the clarification filter and the cell capture filter, the first mixing chamber for containing the backwash liquid.
10. 10. The system of claim 9, further comprising a neutralization buffer reservoir in fluid communication with the first mixing chamber, the first mixing chamber to contain the backwash liquid and neutralization buffer, and to mix the backwash liquid and the neutralization buffer to form a neutralization lysate.
11. 11. The system of claim 10, wherein the clarification filter is in fluid communication with the first mixing chamber, the clarification filter for separating a second waste portion of the biological sample from a target nucleic acid-containing portion.
12. 12. The system of claim 10, wherein the clarification filter comprises the cell capture filter for concentrating cellular components of the biological sample in a first purification step and clarifying the neutralized lysate in a subsequent second purification step.
13. 13. The system of claim 1, wherein the target biomolecule-binding filter of the second biological treatment assembly comprises a silicon-based filter or a microsphere column having an affinity for the target biomolecule.
14. 14. The system of claim 13, wherein the second biological treatment assembly further comprises a purification reagent reservoir in fluid communication with the target biomolecule-binding filter.
15. 15. The system of claim 13, wherein the target biomolecule-binding filter is in fluid communication with the elution buffer reservoir and the output receptacle.
16. 16. The system of claim 15, wherein the second biological treatment assembly comprises a second mixing chamber disposed between the first biological treatment assembly and the target biomolecule-binding filter.
17. 17. The system of claim 16, wherein the second mixing chamber is in fluid communication with an endotoxin removal buffer reservoir.
18. 18. The system of claim 1, wherein the target biomolecule-binding filter employs a nucleic acid binding filter comprising an anion exchange membrane.
19. 19. The system of claim 18, wherein the second biological treatment assembly comprises a precipitation membrane disposed downstream of the anion exchange membrane.
20. 20. The system of claim 19, wherein the anion exchange membrane is used to separate a third waste portion of the biological sample from a target nucleic acid-containing portion.

Description

Systems, methods, and devices for automatically separating nucleic acids from proteins Background Technical Field The present disclosure relates generally to biological sample processing systems, methods, and devices. More particularly, the present disclosure relates to systems, methods, and devices for automated separation of nucleic acids and/or proteins from biological and/or environmental sources. Prior Art Some laboratory procedures remain dominant in execution and these procedures utilize inefficient manual methods, requiring the attention of a scientist or laboratory technician in performing the procedure. Many of these procedures would benefit from automation. For example, nucleic acid purification or isolation protocols, such as large-scale plasmid extraction from bacterial culture, are currently still time-consuming, inefficient, and do not achieve fully automated tasks. Previous attempts to resemble automated guidelines, such as commercial embodiments of the systems disclosed in U.S. patent nos. 8,404,198 and 9,808,799, and similar products, have suffered from numerous drawbacks, including, for example, inoperability when not fully automated and/or when large volumes of samples are available. Gradual improvements such as the introduction of precipitation filters have reduced the required manual operation time. However, even the most advanced nucleic acid purification kit requires a large amount of time investment, for example, endotoxin-free plasmid purification on the maxi, mega or giga scale requires several hours and a separate attention. This is due, at least in part, to the high technical nature of nucleic acid purification and the variety of different tasks that need to be performed during this procedure. For example, many nucleic acid purification protocols flow and route liquids of various viscosities and densities, performed at different times during the purification protocol. Furthermore, during the process of purifying nucleic acids from biological samples, it is important that the buffer solution and reagents be homogeneously mixed with the biological sample and/or filtrate, as this helps to increase the purity and final concentration of the target nucleic acids. Thus, it has proven difficult to incorporate these different liquids into automated processes that provide delayed release, particularly in a manner that allows mixing of the liquids to create a homogeneous solution. Furthermore, it has been demonstrated that providing various buffer solutions and reagents separately in an automated system is both problematic and costly. Ideally, the material containing the various buffer solutions and reagents should be composed of a material that is chemically compatible with the solution being stored (e.g., a material that is not chemically reactive or inert) so that the solution remains functional and active during periods of non-use or storage until such time as the solution is applied for its intended purpose. The variety of filtration steps employed in many nucleic acid purification protocols additionally increase the complexity and difficulty of use in an automated procedure. For example, the various steps in a nucleic acid purification protocol require selective filtration of the solution using mechanical and/or ionic means, followed by washing or purification of the components associated with the filter/filtration membrane. This produces a waste amount several times that of the original biological sample, and the sequestration or disposal of these waste products is a complex factor for automation. In performing in a conventional manner, a technician or scientist performs the nucleic acid purification protocol using a variety of different machines and instruments. This includes, for example, concentrating the biological sample using a centrifuge and a pipette, adding a specific amount of buffer while intermittently stirring or vortexing to homogenize each buffer/reagent inside the solution. Many different filters/filters, columns or magnetic beads are used under centrifugation or vacuum to further develop the nucleic acid purification protocol, and many steps between centrifugation/vacuum steps produce waste products to be disposed of. The above problems are further exacerbated as the volume of the biological sample increases. When processing large volumes of biological samples, more buffers and reagents are typically required, more robust filters, filters and columns. This places more stringent demands on the structural integrity and filtration capacity of the various filters/membranes, which presents challenges in the incorporation and monitoring of such filters/membranes in any automated process. In addition, the use of more buffer and reagents in processing large volumes of biological samples results in more waste. The ability to be responsible for such waste presents an additional unique technical hurdle for any automated process. In addition, since current nucleic acid purifica