EP-4735161-A2 - METHODS AND SYSTEMS FOR AUTOMATED FRACTION SELECTION IN NUCLEIC ACID MANUFACTURING

Abstract

A method for manufacturing nucleic acid molecules, including: obtaining, using a processor, a plurality of fractions from a nucleic acid synthesis procedure; obtaining, using the processor, characterization information regarding each of the plurality of fractions, the characterization information including mass spectrometry and liquid chromatography data for each of the plurality of fractions; identifying, using the processor, a subset of the plurality of fractions to combine to generate a simulated pool based on a metric; simulating, using the processor, a predicted metric for the simulated pool based on identifying the subset of the plurality of fractions to combine; and providing, using the processor, information identifying the subset of fractions to a user to combine into a combined pool based on simulating the predicted metric for the simulated pool.

Inventors

• WANG, Puzhou

Assignees

• Synthego Corporation

Dates

Publication Date

20260506

Application Date

20240614

Claims (1)

1. SYN-028 PATENT Quarles 174774.00178 CLAIMS What is claimed is: 1. A method for manufacturing nucleic acid molecules, comprising: obtaining, using a processor, information identifying a plurality of fractions from a nucleic acid synthesis procedure; obtaining, using the processor, characterization information regarding each of the plurality of fractions, the characterization information comprising liquid chromatography data for each of the plurality of fractions; identifying, using the processor, a subset of the plurality of fractions to combine to generate a simulated pool based on a metric; simulating, using the processor, a predicted metric for the simulated pool based on identifying the subset of the plurality of fractions to combine; and providing, using the processor, information identifying the subset of fractions to a user to combine into a combined pool based on simulating the predicted metric for the simulated pool. 2. The method of claim 1, wherein the liquid chromatography data comprises chromatogram data for each of the plurality of fractions, and wherein simulating a predicted metric for the simulated pool further comprises: aligning the chromatogram data for each of the plurality of fractions, and aggregating the aligned chromatogram data for each of the plurality of fractions based on aligning the chromatogram data to produce simulated pool chromatogram data. 3. The method of claim 2, wherein simulating a predicted metric for the simulated pool further comprises: identifying a plurality of peaks in the simulated pool chromatogram data, determining a main peak in the simulated pool chromatogram data based on identifying the plurality of peaks, and 24 Q B\174774.00178\90537691.1 SYN-028 PATENT Quarles 174774.00178 determining the predicted metric for the simulated pool based on determining the main peak. 4. The method of claim 1, wherein obtaining characterization information further comprises: obtaining characterization information comprising mass spectrometry data for each of the plurality of fractions. 5. The method of claim 4, wherein simulating a predicted metric for the simulated pool further comprises: simulating the predicted metric for the simulated pool based on determining a weighted average of the mass spectrometry data for the subset of the plurality of fractions. 6. The method of claim 5, wherein determining a weighted average of the mass spectrometry data of the subset of the plurality of fractions further comprises: weighting the average of the mass spectrometry data for the subset of the plurality of fractions based on a molarity of nucleic acids in each fraction of the subset of the plurality of fractions. 7. The method of claim 6, wherein simulating a predicted metric for the simulated pool further comprises: generating a predicted mass spectrometry spectrum for the simulated pool. 8. The method of claim 6, wherein simulating a predicted metric for the simulated pool further comprises: simulating a plurality of predicted metrics for the simulated pool, and generating a table of the plurality of predicted metrics. 25 Q B\174774.00178\90537691.1 SYN-028 PATENT Quarles 174774.00178 9. The method of any of the preceding claims, wherein identifying a subset of the plurality of fractions to combine to generate a simulated pool further comprises: adding a fraction to the subset of fractions to generate a new subset of fractions based on determining the weighted average of the metric of the subset of the plurality of fractions, and determining an updated metric for the new subset of the plurality of fractions. 10. The method of claim 1, wherein providing information identifying the subset of fractions to a user to combine into a combined pool further comprises: receiving input from the user selecting a modified subset of fractions to combine into the combined pool, wherein the modified subset of fractions is different from the subset of fractions. 11. The method of claim 1, wherein identifying a subset of the plurality of fractions to combine to generate a simulated pool based on a metric further comprises: identifying a subset of the plurality of fractions to combine to generate a simulated pool based on identifying the simulated pool having a local optimum value. 12. The method of any of the preceding claims, further comprising: combining the subset of fractions into the combined pool. 13. The method of claim 12, further comprising: further processing the combined pool to generate a nucleic acid product. 14. The method of any of the preceding claims, wherein the nucleic acids comprise guide RNA (gRNA) molecules. 15. The method of any of the preceding claims, further comprising: combining a portion of each of the subset of fractions into a mock combined pool, obtaining further characterization information from the mock combined pool, 26 Q B\174774.00178\90537691.1 SYN-028 PATENT Quarles 174774.00178 the further characterization information comprising at least one of liquid chromatography data or mass spectrometry data for the combined pool, determining, based on the further characterization information, whether the mock combined pool satisfies a quality metric for the nucleic acid molecules. 27 Q B\174774.00178\90537691.1

Description

METHODS AND SYSTEMS FOR AUTOMATED FRACTION SELECTION IN NUCLEIC ACID MANUFACTURING CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of priority of U.S. Provisional Application No. 63/524,022, filed on June 29, 2023, which is incorporated herein by reference in its entirety. BACKGROUND In the manufacturing of nucleic acid molecules, such as guide RNA (gRNA) molecules, especially in the large-scale manufacturing, there are common in-line chromatography purification processes that will separate one crude input sample of the oligonucleotides into multiple fractions depending on the interaction strength between chemical species in the sample and chromatography columns used. Each of the resulting fractions will contain various chemical species at different concentrations. To move forward in the production workflow, some of the generated fractions will be combined based on specific constraints on parameters such as purity and yield. Such combination, also known as pooling, of fractions is often irreversible, which emphasizes the critical role of this fraction selection process. To facilitate such fraction selection process, characterization methods such as mass spectrometry (MS) and liquid chromatography (LC) are commonly used to examine the quality of nucleic acid molecules, such as a gRNA molecule, in each fraction. Given chemistry knowledge of gRNA synthesis, algorithms have also been developed to quantify gRNA quality into MS and LC metrics for various fractions. Typically the data are analyzed manually to select which fractions to combine for moving forward. However, with an increasing number of fractions, manual examination of all characterization data, including both the traces and all metrics, quickly turns into a mundane or even impossible task. Consistent quality and yield suffer as a result. Therefore, there is a need for a better fraction selection process to speed up the overall workflow without compromising the quality of the product during the process as well as the final product. Q   B\174774.00178\90537691.1 SYN-028 PATENT Quarles 174774.00178 SUMMARY According to various embodiments, the present disclosure provides apparatus, systems, and methods for manufacturing nucleic acid molecules. One embodiment of the method includes obtaining information identifying a plurality of fractions from a nucleic acid synthesis procedure; obtaining characterization information regarding each of the plurality of fractions, where the characterization information may include mass spectrometry and/or liquid chromatography data for each of the plurality of fractions; identifying a subset of the plurality of fractions to combine to generate a simulated pool based on a metric; simulating a predicted metric for the simulated pool based on identifying the subset of the plurality of fractions to combine; and providing information identifying the subset of fractions to a user to combine into a combined pool based on simulating the predicted metric for the simulated pool. In various embodiments, the apparatus, systems, and methods may be implemented using a processor (e.g., a microprocessor) which may be part of a local or networked computing system and which may be coupled to a non-transient computer-readable medium containing instructions for carrying out the procedures. In some embodiments of the method the liquid chromatography data may include chromatogram data for each of the plurality of fractions, and simulating a predicted metric for the simulated pool may further include aligning the chromatogram data for each of the plurality of fractions, and aggregating the aligned chromatogram data for each of the plurality of fractions based on aligning the chromatogram data to produce simulated pool chromatogram data. In certain embodiments of the method, simulating a predicted metric for the simulated pool may further include identifying a plurality of peaks in the simulated pool chromatogram data, determining a main peak in the simulated pool chromatogram data based on identifying the plurality of peaks, and determining the predicted metric for the simulated pool based on determining the main peak. In particular embodiments of the method, obtaining characterization information may further include obtaining characterization information including mass spectrometry data for each of the plurality of fractions. In various embodiments of the method, simulating a predicted metric for the simulated pool may further include simulating the predicted metric for the simulated pool based on determining a weighted average of the mass spectrometry data for the subset of the 2 Q   B\174774.00178\90537691.1 SYN-028 PATENT Quarles 174774.00178 plurality of fractions. In some embodiments of the method, determining a weighted average of the mass spectrometry data of the subset of the plurality of fractions may further include weighting the average of the mass spectrometry data for the subset of the plural