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ABSTRACT Peptides are widely used in biomaterials due to their ease of synthesis, ability to signal cells, and modify the properties of biomaterials. A key benefit of using peptides is that they are natural substrates for cell‐secreted enzymes, which creates the possibility of utilizing cell‐secreted enzymes for tuning cell–material interactions. However, these enzymes can also induce unwanted degradation of bioactive peptides in biomaterials, or in peptide therapies. Liquid chromatography–mass spectrometry (LC–MS) is a widely used, powerful methodology that can separate complex mixtures of molecules and quantify numerous analytes within a single run. There are several challenges in using LC–MS for the multiplexed quantification of cell‐induced peptide degradation, including the need for nondegradable internal standards and the identification of optimal sample storage conditions. Another problem is that cell culture media and biological samples typically contain both proteins and lipids that can accumulate on chromatography columns and degrade their performance. Removing these constituents can be expensive, time‐consuming, and increases sample variability. However, loading unpurified samples onto the column without removing lipids and proteins will foul the column. Here, we show that directly injecting complex, unpurified samples onto the LC–MS without any purification enables rapid and accurate quantification of peptide concentration and that hundreds of LC–MS runs can be done on a single column without significantly diminishing the ability to quantify the degradation of peptide libraries. To understand how repeated injections degrade column performance, a model library was injected into the LC–MS hundreds of times. It was then determined that column failure is evident when hydrophilic peptides are no longer retained on the column and that failure can be easily identified by using standard peptide mixtures for column benchmarking. In total, this work introduces a simple and effective method for simultaneously quantifying the degradation of dozens of peptides in cell culture. By providing a streamlined and cost‐effective method for the direct quantification of peptide degradation in complex biological samples, this work enables more efficient assessment of peptide stability and functionality, facilitating the development of advanced biomaterials and peptide‐based therapies.
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Rapid metal speciation of cell culture media using reversed‐phase separations and inductively coupled plasma optical emission spectrometry
Abstract Cell culture media metal content is critical in mammalian cell growth and monoclonal antibody productivity. The variability in metal concentrations has multiple sources of origin. As such, there is a need to analyze media before, during, and after production. Furthermore, it is not the simple presence of a given metal that can impact processes, but also their chemical form that is, speciation. To a first approximation, it is instructive to simply and quickly ascertain if the metals exist as inorganic (free metal) ions or are part of an organometallic complex (ligated). Here we present a simple workflow involving the capture of ligated metals on a fiber stationary phase with passage of the free ions to an inductively coupled plasma optical emission spectrometry for quantification; the captured species are subsequently eluted for quantification. This first level of speciation (free vs. ligated) can be informative towards sources of contaminant metal species and means to assess bioreactor processes.
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- Award ID(s):
- 1624641
- PAR ID:
- 10396806
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biotechnology Progress
- Volume:
- 39
- Issue:
- 1
- ISSN:
- 8756-7938
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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