ADD results for biotin QC

tex/thesis.tex

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