ADD results for biotin QC

This commit is contained in:
Nathan Dwarshuis 2021-07-27 17:18:06 -04:00
parent f91a944aa5
commit 8126156630
1 changed files with 65 additions and 3 deletions

View File

@ -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 were removed from the retronectin plates using vigorous pipetting and
transferred to another 96 well plate wherein expansion continued. 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} \subsection{Luminex Analysis}
Luminex was performed using a \product{ProcartaPlex kit}{\thermo}{custom} for 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 biotin input (which corresponded to \SI{2.5}{\nmol\per\mg\of{\dms}}) thus we
used this in subsequent processes. 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) % TODO flip the rows of this figure (right now the text is backward)
\begin{figure*}[ht!] \begin{figure*}[ht!]
\begingroup \begingroup
@ -1061,6 +1061,67 @@ used this in subsequent processes.
\input{../tables/carrier_properties.tex} \input{../tables/carrier_properties.tex}
\end{table} \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!] \begin{figure*}[ht!]
\begingroup \begingroup
@ -1087,6 +1148,7 @@ used this in subsequent processes.
\label{fig:dms_flowchart} \label{fig:dms_flowchart}
\end{figure*} \end{figure*}
% RESULT add paragraph explaining the reaction kinetics stuff
\begin{figure*}[ht!] \begin{figure*}[ht!]
\begingroup \begingroup