Biomedical Engineering Reference
In-Depth Information
performed flow injection electrospray mass spectrometry on feed media and cell culture
fluid and correlated an increase in a “marker”mass with the lack of a trace element needed
by the cells. The mass correlated with a buildup of lactate, which is a known metabolic
dead-end in the Krebs cycle. By determining the identification of the marker mass, they
were able to isolate a portion of theKrebs cycle that relied on the presence of a certain trace
element to keep the metabolic process from shunting to an anaerobic pathway. By adding
more of the trace element copper (Cu
þþ
) to the process, the overall improvement cell
viability was realized.
Other tools used to assess complex raw materials can range from the simplistic
fingerprinting approach to a complete breakdown of each component by multiple
techniques. Hydrolysates from various animal and plant sources are regularly used in
cell culture processes. A simple approach to assess hydrolysates is running reverse-phase
HPLC with a diode array detector on a C-18 column and creating hydrolysate finger-
prints from the spectra. The spectra from source material deemed “good” from use
testing can be compared with incoming hydrolysates using pattern-matching software to
elucidate any differences. Layers of informationmay be supplemented by using detectors
in series, such as mass spectrometry, to identify the exact nature of the difference.
Still other raw materials require a complete breakdown to understand how multiple
components can interact to affect cell growth. Wei and Lanan (data unpublished, [54])
used induction coupled plasmamass spectrometry, NMR, diode array HPLC spectra, and
mass spectrometry coupled with chemometrics such as principal components analysis
and PLS-based visualization tools to demonstrate how components in hydrolysates such
as trace elements, hormone analogues, and key peptide quantities impacted cell viability.
They were able to determine by ICP-MS the varying levels of the trace element zinc in
different lots of hydrolysate, but this component alone was not responsible for all of the
variability. They used NMR and diode array HPLC spectra to further determine the
presence of certain hormone analogues and peptides in each lot of hydrolysate. By
putting the data together and ranking combinations of each component based on effect on
cell viability, they were able to put together a matrix of component interactions that
described how each component and combinations of components were correlated with
cell growth.
With the ability of rawmaterials to have the largest impact on process variability, it is
becoming increasingly important to gain a deeper fundamental understanding of how
each component influences critical process parameters and product quality attributes
including post-translational modifications such as glycosylation. As raw materials
become more defined and characterized, the ability to selectively influence product
quality attributes during the course of a bioreactor run becomes possible. NIR can also be
used for resin identity as a quality check before column-packing procedures are initiated.
12.6 CHEMOMETRICS AND ADVANCED PROCESS
CONTROL TOOLS
Aforementioned sampling techniques, new sensor technologies, and new analyzers
generate complex data that require special treatment to extract the embedded useful
