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ENH integrate the kinetics figure

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Nathan Dwarshuis 2021-07-31 20:11:40 -04:00
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@ -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