From 81261566304ebb8ef3b2dc18f96cdf8f0ca7ee06 Mon Sep 17 00:00:00 2001 From: ndwarshuis Date: Tue, 27 Jul 2021 17:18:06 -0400 Subject: [PATCH] ADD results for biotin QC --- tex/thesis.tex | 68 +++++++++++++++++++++++++++++++++++++++++++++++--- 1 file changed, 65 insertions(+), 3 deletions(-) diff --git a/tex/thesis.tex b/tex/thesis.tex index 371f943..e0b4aee 100644 --- a/tex/thesis.tex +++ b/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