ADD results for biotin QC
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@ -948,6 +948,9 @@ plates and incubated for another \SI{24}{\hour}. Cells and beads/\glspl{dms}
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were removed from the retronectin plates using vigorous pipetting and
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were removed from the retronectin plates using vigorous pipetting and
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transferred to another 96 well plate wherein expansion continued.
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transferred to another 96 well plate wherein expansion continued.
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% METHOD snb decay curve generation and analysis (including the equation used to
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% fit the data)
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\subsection{Luminex Analysis}
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\subsection{Luminex Analysis}
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Luminex was performed using a \product{ProcartaPlex kit}{\thermo}{custom} for
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Luminex was performed using a \product{ProcartaPlex kit}{\thermo}{custom} for
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@ -1022,9 +1025,6 @@ noted that the maximal \gls{mab} binding capacity occurred near \SI{50}{\nmol}
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biotin input (which corresponded to \SI{2.5}{\nmol\per\mg\of{\dms}}) thus we
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biotin input (which corresponded to \SI{2.5}{\nmol\per\mg\of{\dms}}) thus we
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used this in subsequent processes.
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used this in subsequent processes.
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% RESULT add paragraph explaining the qc stuff
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% RESULT add paragraph explaining the reaction kinetics stuff
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% TODO flip the rows of this figure (right now the text is backward)
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% TODO flip the rows of this figure (right now the text is backward)
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\begin{figure*}[ht!]
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\begin{figure*}[ht!]
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\begingroup
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\begingroup
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@ -1061,6 +1061,67 @@ used this in subsequent processes.
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\input{../tables/carrier_properties.tex}
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\input{../tables/carrier_properties.tex}
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\end{table}
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\end{table}
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% TODO add chemical equation for which reactions I am describing here
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We then asked how sensitive the \gls{dms} manufacturing process was to a variety
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of variables. In particular, we focused on the biotin-binding step, since it
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appeared that the \gls{mab} binding was quadratically related to biotin binding
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(\cref{fig:mab_coating}) and thus controlling the biotin binding step would be
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critical to controlling the amount and \glspl{mab} and thus the amount of signal
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the T cells receive downstream.
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To answer this question, we first performed a \gls{doe} to understand the effect
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of reaction parameters on biotin binding. The parameters included in this
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\gls{doe} were temperature, microcarrier mass, and \gls{snb} input mass. These
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were parameters that we specifically controlled but hypothesized might have some
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sensitivity on the final biotin mass attachment rate depending on their noise
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and uncertainty. In particular, temperature was `controlled' only by allowing
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the microcarrier suspension to come to \gls{rt} after autoclaving. After
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performing a full factorial \gls{doe} with three center points as the target
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reaction conditions, we found that the final biotin binding mass is only highly
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dependent on biotin input concentration (\cref{fig:dms_qc_doe}). Overall,
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temperature had no effect and carrier mass had no effect at higher temperatures,
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but carrier mass had a slightly positive effect when temperature was low. This
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could be because lower temperature might make spontaneous decay of \gls{snb}
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occur slower, which would give \gls{snb} molecule more opportunity to diffuse
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into the microcarriers and react with amine groups to form attachments. It seems
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that concentration only has a linear effect and doesn't interact with any of the
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other variables, which is not surprisingly given the behavior observed in
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(\cref{fig:biotin_coating})
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We also observed that the reaction pH does not influence the amount of biotin
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attached (\cref{fig:dms_qc_ph}), which indicates that while higher pH might
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increase the number of deprotonated amines on the surface of the microcarrier,
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it also increases the number of OH\textsuperscript{-} groups which can
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spontaneously hydrolyze the \gls{snb} in solution.
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Furthermore, we observed that washing the microcarriers after autoclaving
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increases the biotin binding rate (\cref{fig:dms_qc_washes}). While we did not
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explore this further, one possible explanation for this behavior is that the
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microcarriers have some loose protein in the form of powder or soluble peptides
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that competes for \gls{snb} binding against the surface of the microcarriers,
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and when measuring the supernatent using the \gls{haba} assay, these soluble or
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lightly-suspended peptides/protein fragments are also measured and therefore
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inflate the readout.
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% TABLE decay curve half lives
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Lastly, we asked what the effect on reaction pH had on spontaneous degradation
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of \gls{snb} while in solution. If the \gls{snb} significantly degrades within
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minutes of preparation, then it is important to carefully control the timing
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between \gls{snb} solution preparation and addition to the microcarriers. When
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buffering \gls{pbs} to different pH's, analyzing the decay curves using UV plate
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reader, and fitting an exponential decay equation to the data, we observed that
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the half-life of \gls{snb} in solution decreases
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(\cref{fig:dms_snb_decay_curves}). However, these half-lives are large enough
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(on the order of several hours) not to be of concern assuming that the \gls{snb}
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solution is added within a few minutes of preparation (which it was in all our
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cases). Furthermore, we dissolved our \gls{snb} in \gls{di} water and not
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\gls{pbs} which means the pH is even lower and thus the half life is even
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higher, further showing that the decay of \gls{snb} is not a concern.
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% TODO add the water curve to the figure just to make it clear this is not a
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% concern
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\begin{figure*}[ht!]
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\begin{figure*}[ht!]
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\begingroup
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\begingroup
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@ -1087,6 +1148,7 @@ used this in subsequent processes.
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\label{fig:dms_flowchart}
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\label{fig:dms_flowchart}
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\end{figure*}
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\end{figure*}
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% RESULT add paragraph explaining the reaction kinetics stuff
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\begin{figure*}[ht!]
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\begin{figure*}[ht!]
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\begingroup
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\begingroup
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