Quantifying Sub-visible Particulates in Cell Culture via Lysis and Filtration
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Solution Overview
Problem
Current methods for assessing sub-visible particulates in injectable drug products are not applicable to cell therapies, as cells can be considered particulates, leading to a need for quantifying non-cell sub-visible particulates in cell cultures, which is crucial for ensuring safety in therapeutic cell cultures.
Innovation Solution
The methods involve breaking down cells in a cell culture using lysis, followed by filtration and microscopic analysis to detect and quantify non-cell sub-visible particulates, allowing for the determination of their presence and concentration, even when they are similar in size to cells, thereby addressing the challenge of microcarriers and fines in bioreactor-produced cell cultures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If cells are analyzed directly in cell culture using standard particulate assessment methods, then the assessment can be performed, but cells are incorrectly identified as particulates leading to inaccurate quantification
Solution Approach 1:
The patent extracts and removes cells from the cell culture sample through lysis or filtration before particulate analysis. This separation allows the subsequent detection and quantification of non-cell particulates without interference from intact cells, resolving the measurement accuracy issue.
Solution Approach 2:
The analysis process is segmented into distinct stages: (1) cell removal/lysis, (2) filtration, and (3) particulate detection. This segmentation allows each step to be optimized independently, with cell lysis methods specifically designed to break down cellular structures while preserving non-cell particulates for accurate quantification.
2Productivity
If microcarriers are used to mass-produce adherent cells in bioreactors, then large amounts of cells can be produced, but microcarrier fines remain in the cell culture causing potential injury to recipients
Solution Approach 1:
The patent applies preliminary action by performing cell lysis and filtration steps before final cell therapy formulation. This preliminary processing removes microcarrier fines and other non-cell particulates from the cell culture, preventing potential injury to recipients while preserving the therapeutic cells.
Solution Approach 2:
The patent converts the harmful presence of microcarrier fines into a detectable signal by using the lysis/filtration approach. The fines that would otherwise be hidden among intact cells are now concentrated and visible in the filtrate, allowing for their quantification and removal, thus transforming a contamination problem into a solvable measurement and purification task.
3Measurement precision
If sub-visible particulates are similar in size to cells, then they are difficult to distinguish, but accurate detection is necessary for safety
Solution Approach 1:
The patent extracts cells from the mixture through lysis or filtration, removing the size-based confusion. By eliminating intact cells from the sample, the detection system can focus solely on non-cell particulates regardless of their size, enabling accurate detection of sub-visible particulates that would otherwise be indistinguishable from cells.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
These methods enable the detection and quantification of sub-visible particulates in cell cultures, ensuring the safety and quality of therapeutic cell products by identifying and extrapolating particulate counts across cell culture lots, thereby reducing the risk of injury from foreign particulates.
Implementation Method 1
In certain embodiments, the cells in a cell culture to be analyzed using the methods provided herein are lysed using a solution (e.g., a lysis buffer) that comprises one or more detergents
Data Source
AI summary
Provided are methods for quantifying and/or detecting sub-visible particulates in cell cultures. Specifically, the methods comprise a step of breaking down, e.g., lysing, cells in a cell culture. The methods can further comprising filtering the cell culture through a filter. Further provided are methods of quantifying sub-visible particulates that do not pass through the filter using a microscope.