US-12625041-B2 - Automated sample preparation for spent media analysis

US12625041B2US 12625041 B2US12625041 B2US 12625041B2US-12625041-B2

Abstract

A method for processing a sample of a cell culture composition includes diluting the sample followed by filtering the diluted sample, wherein the dilution may be at least 10 parts by volume of a diluent to one part sample, and the filter may be a porous membrane with a molecular weight cutoff of less than 20,000 Daltons. The dilution may be carried out at a multiple-port dilution valve having a first condition and a second condition. The sample may be mixed with the diluent at a predetermined dilution ratio when the dilution valve is in the second condition.

Inventors

Quan Liu
John Barnacle
Eric Beemer

Assignees

IDEX HEALTH & SCIENCE LLC

Dates

Publication Date: 20260512
Application Date: 20221215

Claims (13)

1 . A method for processing a cell culture composition of cells and spent media, the method comprising: (a) obtaining a sample of the cell culture composition from a source; (b) providing a processing apparatus having: (i) a dilution module for mixing the sample of the cell culture composition with a diluent to form a dilution composition, wherein the dilution module is operable in at least two conditions, with a first load condition loading the cell culture composition into a sample loading chamber, and a second inject condition combining the cell culture composition from the sample loading chamber with the diluent to form the dilution composition; (ii) a filtration module having a filter for filtering the dilution composition into a filtrate and a retentate; (iii) a sample collector for distinguishing a filtrate sample from the filtrate, wherein the processing apparatus defines a first fluidic pathway from the dilution module, through the filtration module, and to the sample collector; (c) motivating the dilution composition through the first fluidic pathway; and (d) operating the sample collector to: (i) begin distinguishing the filtrate sample from the filtrate at a first steady state time point when a concentration of the sample in the filtrate at the sample collector has increased to a first steady state concentration; and (ii) subsequent to step (d) (i), cease distinguishing the filtrate sample from the filtrate by no later than a second steady state time point when the concentration of the sample in the filtrate at the sample collector has decreased from the first steady state concentration by a predetermined threshold value.
2 . The method as in claim 1 wherein the predetermined threshold value is 10%.
3 . The method as in claim 1 wherein the sample collector distinguishes the filtrate sample from the filtrate by separating the filtrate sample from the filtrate.
4 . The method as in claim 3 , including motivating the filtrate sample to an analysis module for analyzing the filtrate sample.
5 . The method as in claim 4 wherein the analysis module includes a liquid chromatograph, a mass spectrometer, and combinations thereof.
6 . The method as in claim 1 , including operating the dilution module to mix the sample with the diluent at a mixing ratio of at least 10 parts by volume of the diluent to one part by volume of the sample to form the dilution composition.
7 . The method as in claim 6 wherein the sample of the cell culture matrix is mixed with the diluent without undergoing a precipitation process prior to the mixing.
8 . The method as in claim 7 wherein the sample of the cell culture matrix is mixed with the diluent without undergoing a centrifugation process prior to the mixing.
9 . The method as in claim 1 , including recording a first switch time point when the dilution module switches from the first condition to the second condition, and recording a second switch time point when the dilution module switches from the second condition to the first condition.
10 . The method as in claim 9 , including measuring a first delay time period between the first switch time point and the first steady state time point that is subsequent to the first switch time point, and measuring a second delay time period between the first steady state time point and the second steady state time point that is subsequent to the first steady state time point.
11 . The method as in claim 10 , including operating the sample collector to begin distinguishing the filtrate sample from the filtrate upon an expiration of the first delay time period from the first switch point, and to cease distinguishing the filtrate sample from the filtrate no later than the expiration of the second delay period from the first steady state time point.
12 . The method as in claim 11 , including operating the sample collector to cease distinguishing the filtrate sample from the filtrate upon the expiration of the second delay time period from the first steady state time point.
13 . The method as in claim 11 , including measuring a third delay time period between the second steady state time point and a third steady state time point subsequent to the second steady state time point when the concentration of sample in the filtrate has decreased to a second steady state concentration.

Description

FIELD OF THE INVENTION The present invention relates to processing of biological samples generally, and more particularly to an automated system for preparing samples of spent cell culture compositions for analytical analysis. BACKGROUND OF THE INVENTION Cell culture is one of the major techniques in the life sciences. The most important and crucial step in cell culture is selecting appropriate growth medium for in vitro cultivation. A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms, cells, or small plants. Cell culture media generally comprise an appropriate source of energy and compounds which regulate the cell cycle. A typical culture medium is composed of a complement of amino acids, vitamins, inorganic salts, glucose, and serum as a source of growth factors, hormones, and attachment factors. The development of cell culture medium is critical for cell culture. Spent media analysis is an important tool used to provide crucial information about the amino acids and nutrients that cells use during their growth phase. Spent media analysis also provides details about the metabolites that are produced as a result of cell growth. This is useful to cell line and process development teams, in that it provides data on media composition performance, optimal harvest time and product quality changes that can occur during culture. For example, investigators may be interested in monitoring daily changes in amino acids and metabolites to optimize growth conditions for clones, promote high cell viability, maximize production of titer, ensure high product quality, and reduce or eliminate sequence variant misincorporations. It becomes an important Critical Process Parameter (CPP) that needs to be monitored frequently. The existing workflow for analyzing spent media includes mainly manual steps that are susceptible to human error. Current spent media sample preparation workflows lack efficiency and reliability. The present invention addresses the need to automate the sample preparation process to improve throughput and to facilitate liquid chromatography/mass spectrometry sample analyzation. An example of currently existing workflow for sample preparation is illustrated in FIG. 1, and starts with collecting sample from a bioreactor. The sample taken out of the bioreactor normally has solid cells and/or cell debris. The first step of sample preparation is to remove the solid cells and cell debris. The common practice is to use centrifugation to separate solid from liquid in the sample, and thereafter to collect the supernatant from the centrifuge for the next phase. After the cells/cell debris are removed in the initial centrifugation step, the liquid phase typically still contains large protein molecules. For cell culture/spent media analysis, large protein molecules can pose challenges for liquid chromatography/mass spectrometry analysis. The large protein molecules can become stuck inside the column, reduce column life and increase the back pressure through the column. The protein molecules also compete with ionization, which reduces the effectiveness of the mass spectrometry ionization process. The common practice to remove/reduce large protein molecules from the liquid phase of the sample is through chemical precipitation, followed by centrifugation or filtration. This step is primarily a manual operation, and typically requires that the sample be diluted 2×-5× by adding acetonitrile or methanol. After the large protein molecules are removed, the sample is diluted to a level that is appropriate for LC/MS analysis. The dilution level for this step could be between 10×-100×. The total dilution level could be up to 200×-300×. The existing workflow for preparing spent media samples contains mainly manual operations, including multi-dilution processes, loading centrifuges, and removing supernatant out from the mixture. With a large quantity of sample to process, the sample preparation can become a bottleneck. This invention automates the process to shorten the sample preparation time and to bring better results to the final analysis. SUMMARY OF THE INVENTION By means of the present invention, sample dilution and optionally filtration may be accomplished with highly controllable automation tools that increases both dilution efficiency and accuracy. The system of the present invention further enables controllability in dilution ratios for mixing multiple samples. In an embodiment involving a cell culture composition of cells and spent media, a method for processing the composition includes providing a processing apparatus having a dilution module for mixing a sample of the composition with a diluent to form a dilution composition, a filtration module having a filter for filtering the dilution composition into a filtrate and a retentate, and a sample collector for distinguishing a filtrate sample from the filtrate. The dilution module may be operable in at least two conditions