diff --git a/tex/thesis.tex b/tex/thesis.tex index c99548e..2ccec6e 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -1255,6 +1255,32 @@ appeared that the \gls{mab} binding was quadratically related to biotin binding critical to controlling the amount and \glspl{mab} and thus the amount of signal the T cells receive downstream. +\begin{figure*}[ht!] + \begingroup + + \includegraphics{../figures/dms_qc.png} + \phantomsubcaption\label{fig:dms_qc_doe} + \phantomsubcaption\label{fig:dms_qc_ph} + \phantomsubcaption\label{fig:dms_qc_washes} + \phantomsubcaption\label{fig:dms_snb_decay_curves} + + \endgroup + \caption[\gls{dms} Quality Control] + {\gls{dms} quality control investigation and development + \subcap{fig:dms_qc_doe}{\gls{doe} investigating the effect of initial mass + of microcarriers, reaction temperature, and biotin concentration on + biotin attachment.} + \subcap{fig:dms_qc_ph}{Effect of reaction ph on biotin attachment.} + \subcap{fig:dms_qc_washes}{effect of post-autoclave washing of the + microcarriers on biotin attachment.} + \subcap{fig:dms_snb_decay_curves}{Hydrolysis curves of \gls{snb} in + \gls{pbs} or \gls{di} water.} + All statistical tests where p-values are noted are given by two-tailed t + tests. + } + \label{fig:dms_flowchart} +\end{figure*} + 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 @@ -1305,64 +1331,6 @@ to the microcarrier suspension (which itself is in \gls{pbs}) this result indicated that hydrolysis is not of concern when adding \gls{snb} within minutes. -\begin{figure*}[ht!] - \begingroup - - \includegraphics{../figures/dms_qc.png} - \phantomsubcaption\label{fig:dms_qc_doe} - \phantomsubcaption\label{fig:dms_qc_ph} - \phantomsubcaption\label{fig:dms_qc_washes} - \phantomsubcaption\label{fig:dms_snb_decay_curves} - - \endgroup - \caption[\gls{dms} Quality Control] - {\gls{dms} quality control investigation and development - \subcap{fig:dms_qc_doe}{\gls{doe} investigating the effect of initial mass - of microcarriers, reaction temperature, and biotin concentration on - biotin attachment.} - \subcap{fig:dms_qc_ph}{Effect of reaction ph on biotin attachment.} - \subcap{fig:dms_qc_washes}{effect of post-autoclave washing of the - microcarriers on biotin attachment.} - \subcap{fig:dms_snb_decay_curves}{Hydrolysis curves of \gls{snb} in - \gls{pbs} or \gls{di} water.} - All statistical tests where p-values are noted are given by two-tailed t - tests. - } - \label{fig:dms_flowchart} -\end{figure*} - -We also investigated the reaction kinetics of all three coating steps. - -To quantify the reaction kinetics of the biotin binding step, we reacted -multiple batches of \SI{20}{\mg\per\ml} microcarriers in \gls{pbs} at \gls{rt} -with \gls{snb} in parallel and sacrificially analyzed each at varying timepoints -using the \gls{haba} assay. This was performed at two different concentrations. -We observed that for either concentration, the reaction was over in -\SIrange{20}{30}{\minute} (\cref{fig:dms_biotin_rxn_mass}). Furthermore, when -put in terms of fraction of input \gls{snb}, we observed that the curves are -almost identical (\cref{fig:dms_biotin_rxn_frac}). Given this, the reaction step -for biotin attached was set to \SI{30}{\minute}. - -% TODO these numbers might be totally incorrect -Next, we quantified the amount of \gls{stp} reacted with the surface of the -biotin-coated microcarriers. Different batches of biotin-coated \glspl{dms} were -coated with \SI{40}{\ug\per\ml} \gls{stp} and sampled at various timepoints -using the \gls{bca} assay to indirectly quantify the amount of attached -\gls{stp} mass. We found this reaction took \SI{45}{\minute} -(\cref{fig:dms_stp_per_time}). - -% TODO find real numbers for this -Finally, we used the reaction data from the \gls{stp} binding curve to estimate -the \gls{mab} binding curve. Assuming a quasi-steady-state paradigm, we -estimated that the diffusion rate coefficient for the microcarriers was -{\#}{diffusion rate}. Using this diffusion rate and the maximum mass of -\glspl{mab} bound the microcarriers (\cref{fig:mab_coating}), we estimated that -the \gls{mab} reaction should proceed in {\#}{mab curve}. - -% TODO add additional paragraph about how this diffusion coefficient was used to -% estimate the wash step times. - -% RESULT talk about the kinetic stuff in this figure more \begin{figure*}[ht!] \begingroup @@ -1387,6 +1355,64 @@ the \gls{mab} reaction should proceed in {\#}{mab curve}. \label{fig:dms_kinetics} \end{figure*} +We also investigated the reaction kinetics of all three coating steps. + +To quantify the reaction kinetics of the biotin binding step, we reacted +multiple batches of \SI{20}{\mg\per\ml} microcarriers in \gls{pbs} at \gls{rt} +with \gls{snb} in parallel and sacrificially analyzed each at varying timepoints +using the \gls{haba} assay. This was performed at two different concentrations. +We observed that for either concentration, the reaction was over in +\SIrange{20}{30}{\minute} (\cref{fig:dms_biotin_rxn_mass}). Furthermore, when +put in terms of fraction of input \gls{snb}, we observed that the curves are +almost identical (\cref{fig:dms_biotin_rxn_frac}). Given this, the reaction step +for biotin attached was set to \SI{30}{\minute}. + +% TODO these numbers might be totally incorrect +% TODO state what the effective diffusivity is +Next, we quantified the amount of \gls{stp} reacted with the surface of the +biotin-coated microcarriers. Different batches of biotin-coated \glspl{dms} were +coated with \SI{40}{\ug\per\ml} \gls{stp} and sampled at intermediate timepoints +using the \gls{bca} assay to indirectly quantify the amount of attached +\gls{stp} mass. We found this reaction took approximately \SI{30}{\minute} +(\cref{fig:dms_stp_per_time}). Assuming a quasi-steady-state paradigm, we used +this experimental binding data to fit a continuous model for the \gls{stp} +binding reaction. Using the diffusion rate of the \gls{stp}, we then calculated +the effective diffusivity of the microcarriers to be {\#}. + +Using this effective diffusivity and the known diffusion coefficient of a +\gls{mab} protein in water, we calculated predict the binding of \glspl{mab} per +time onto the microcarriers (this obviously assumes that the effectively +diffusivity is independent of the protein used, which should be reasonable given +that the pores of the microcarriers are huge compared to the proteins, and we +don't expect any significant reaction between the protein and the microcarrier +surface save for the \gls{stp}-biotin binding reaction). According to this +model, the \gls{mab} binding reaction should be complete within \SI{15}{\minute} +under the conditions used for our protocol (\cref{fig:dms_mab_per_time}). Note +that our unoptimized coated steps were done in \SI{45}{\minute}, which seemed +reasonable given the slightly larger hydrodynamic radius of \glspl{mab} compared +to \gls{stp} which was shown to react in \SI{30}{\minutes} experimentally. The +results of this model should be experimentally verified. + +% TODO find the actual numbers for this +Finally, we used the effective diffusivity of the microcarriers to predict the +time needed for wash steps. This is important, as failing to wash out residual +free \gls{snb} (for example) could occupy binding sites on the \gls{stp} +molecules, lowering the effective binding capacity of the \gls{mab} downstream. +Once again, we assumed the microcarriers to be porous spheres, this time with an +initial concentration of \gls{snb}, \gls{stp}, or \glspl{mab} equal to the final +concentration of the bulk concentration of the previous binding step, and +calculated the amount of time it would take for the concentration profile inside +the microcarriers to equilibrate to the bulk in the wash step. Using this model, +we found that the wash times for \gls{snb}, \gls{stp}, and \glspl{mab} was +\SI{10}{\minute}, {\#} minutes, and {\#} minutes respectively. Note that +\gls{snb}, \gls{stp}, and \glspl{mab} each required 3, 2, and 2 washes to reduce +the concentration down to a level that was 1/1000 of the starting concentration +(which was deemed to be acceptable for preventing downstream inhibition). Using +this in our protocol, we verified that the \gls{snb} was totally undetectable +after washing (\cref{fig:dms_biotin_washed}). The other two species need to be +verified, but note that the consequences of residual \gls{stp} or \gls{mab} are +far less severe than that of \gls{snb}. + \subsection{DMSs can efficiently expand T cells compared to beads} % RESULT add other subfigures here