ADD section on reducing noise

tex/thesis.tex

@@ -241,6 +241,7 @@
 \newacronym{hepes}{HEPES}{4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid}
 \newacronym{nhs}{NHS}{N-hydroxysulfosuccinimide}
 \newacronym{tocsy}{TOCSY}{total correlation spectroscopy}
+\newacronym{hplc}{HPLC}{high-performance liquid chromatography}
 
 %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
 % SI units for uber nerds
@@ -4546,27 +4547,27 @@ serve as a drop-in substitution to provide these benefits.
 There are several important next steps to perform with this work, many of which
 will be relevent to using this technology in a clinical trial:
 
-\subsection{Translation to GMP Process}
+\subsection{Using GMP Materials}
 
 While this work was done with translatability and \gls{qc} in mind, an important
 feature that is missing from the process currently is the use of \gls{gmp}
-methods and materials. The microcarriers themselves are made from
-porcine-derived collagen, which itself is not \gls{gmp}-compliant due to its
-non-human animal origins. However, using any other source of collagen should
-work so long as the structure of the microcarriers remains relatively similar
-and it has lysine groups that can react with the \gls{snb} to attach \gls{stp}
-and \glspl{mab}. Obviously these would need to be tested and verified, but these
-should not be insurmountable. Furthermore, the \gls{mab} binding step requires
-\gls{bsa} to prevent adsorption to the non-polar polymer walls of the reaction
-tubes. A human carrier protein such as \gls{hsa} could be used in its place to
-eliminate the non-human animal origin material, but this could be much more
-expensive. Alternatively, the use of protein could be replaced altogether by a
-non-ionic detergent such as Tween-20 or Tween-80, which are already used for
-commercial \gls{mab} formulations for precisely this purpose\cite{Kerwin2008}.
-Validating the process with Tween would be the best next step to eliminate
-\gls{bsa} from the process. The \gls{stp} and \glspl{mab} in this work were
-not \gls{gmp}-grade; however, they are commonly used in clinical technology such
-as dynabeads and thus the research-grade proteins used here could be easily
+materials. The microcarriers themselves are made from porcine-derived collagen,
+which itself is not \gls{gmp}-compliant due to its non-human animal origins.
+However, using any other source of collagen should work so long as the structure
+of the microcarriers remains relatively similar and it has lysine groups that
+can react with the \gls{snb} to attach \gls{stp} and \glspl{mab}. Obviously
+these would need to be tested and verified, but these should not be
+insurmountable. Furthermore, the \gls{mab} binding step requires \gls{bsa} to
+prevent adsorption to the non-polar polymer walls of the reaction tubes. A human
+carrier protein such as \gls{hsa} could be used in its place to eliminate the
+non-human animal origin material, but this could be much more expensive.
+Alternatively, the use of protein could be replaced altogether by a non-ionic
+detergent such as Tween-20 or Tween-80, which are already used for commercial
+\gls{mab} formulations for precisely this purpose\cite{Kerwin2008}. Validating
+the process with Tween would be the best next step to eliminate \gls{bsa} from
+the process. The \gls{stp} and \glspl{mab} in this work were not
+\gls{gmp}-grade; however, they are commonly used in clinical technology such as
+dynabeads and thus the research-grade proteins used here could be easily
 replaced. The \gls{snb} is a synthetic small molecule and thus does not have any
 animal-origin concerns.
 
@@ -4613,11 +4614,11 @@ to an asymmetric cytokine cross-talk which accounts for the population-level
 differences seen in comparison to the beads.
 
 Experimentally, the first step involves separating the \glspl{dms} from the
-loosely or non-adhered T cells and digesting the \glspl{dms} wth \gls{cold}
+loosely or non-adhered T cells and digesting the \glspl{dms} with \gls{cold}
 (concentrations of \SI{10}{\ug\per\ml} will completely the \glspl{dms} within
 \SIrange{30}{45}{\min}) isolate the interior T cells. Unfortunately, only
 \SIrange{10}{20}{\percent} of all cells will be on the interior, so the interior
-group may only have cells on the order of \si{1e3} to \si{1e4} for analysis. A
+group may only have cells on the order of \num{1e3} to \num{1e4} for analysis. A
 good first pass experiment would be to analyze both populations with a T cell
 differentiation/activation state flow panel first (since flow cytometry is
 relatively cheap and doesn't require a large number of cells) to simply
@@ -4663,6 +4664,90 @@ interacting with the bulk material itself, the void fraction and pore size will
 need to be taken into account to find the bulk material properties of the
 cross-linked gelatin\cite{Wang1984}.
 
+% TODO this might warrant a better figure
+\subsection{Reducing Ligand Variance}
+
+While we have robust quality control steps to quantify each step of the
+\gls{dms} coating process, we still see high variance across time and personnel.
+This is less than ideal for translation.
+
+When investigating the \gls{mab} and \gls{stp} binding, it appears that there is
+a significant variance both between and within different experiments (even
+within the same operator).  The following are a list of variance sources and
+potential mitigation strategies:
+
+\begin{description}
+\item[Mass loss during autoclaving --] In order to ensure a consistent reaction
+  volume, we mass the tube after adding carriers and \gls{pbs} prior to
+  autoclaving. Autoclaving and washing will cause variations in the liquid
+  level, and these are corrected using the pre-recorded tube mass. However, this
+  assumes that the mass of the tube never changes, which may or may not be true
+  in an autoclave where the temperature easily causes deformation of the plastic
+  tube material. This can easily be tested by autoclaving empty tubes and
+  observing a mass change. If there is a mass change, it may be mitigated by
+  pre-autoclaving tubes (assuming that autoclaving is idempotent with respect to
+  mass loss), or alternatively we could estimate the bias by autoclaving a
+  set of tubes, recording the mean mass loss, and using this to correct the tube
+  mass for downstream calculations.
+\item[Errors in initial microcarrier massing --] The massing of microcarriers at
+  the very beginning of the process requires care due to the low target mass and
+  the propensity for both the plastic tubes and microcarriers to accumulate
+  static. Oddly, the biotin attachment readout does not seem to be much affected
+  by the mass of carriers (\cref{fig:dms_qc_doe}); however, this merely means
+  that errors in carrier mass lead to different biotin surface densities, which
+  downstream causes different ratios of \gls{stp} and \gls{mab} attachment since
+  these relationships are non-linear with respect to biotin surface density
+  (\cref{fig:stp_coating,fig:mab_coating}) (this is in addition to the fact that
+  having more or less carriers will bias the total amount of \gls{stp} and
+  \gls{mab} able to bind). A quick survey of operators revealed that acceptable
+  margins for error in mass range from \SIrange{2.5}{5.0}{\percent} (eg, a
+  target value $X$ \si{\mg} will be accepted as $X$ at plus or minus these
+  margins). These could easily be reduced and standardized via protocol.
+  Additionally, we do not currently record the exact mass of microcarriers
+  weighed for each batch. Knowing this would allow us to pinpoint how much of
+  this variance is due to our acceptable measurement margins and what errors may
+  arise from static and other instrument noise.
+\item[Centrifugation after washing --] After coating the \gls{dms} with \gls{snb},
+  \gls{stp}, or \glspl{mab}, they must be washed. After washing, they must be
+  massed in order to ensure the reaction volume is consistent. Ideally, the
+  tubes are centrifuged after washing to ensure that all liquid is at the bottom
+  prior to beginning the next coating step. Upon survey, not all operators
+  follow this protocol, and the protocols are not written such to make this
+  obvious. Therefore, protocols will be revised followed by additional training.
+\item[Accidental microcarrier removal --] When washing the microcarriers after a
+  coating step, liquid is aspirated using a stripette. The carriers should be at
+  the bottom of the tube during this aspiration step. Depending on the skill and
+  care of the operator, carriers may be aspirated with the liquid during this
+  step. If this happens, downstream \gls{qc} assays will not reflect the true
+  binding magnitude, as these assays assume the number of carriers is constant.
+\item[\gls{bsa} binding kinetics --] Prior to \gls{mab} addition, \gls{bsa} is
+  added to the \gls{mab} to block binding to the tubes. \glspl{mab} are added
+  immediately after adding the \gls{bsa}, which means the \gls{bsa} has almost
+  no time to mix completely and thus the \gls{mab} could come into contact with
+  the sides of the tube unshielded. In theory this could cause the \gls{mab}
+  reading to be lower on the \gls{elisa} during \gls{qc}. This problem may be
+  minor since significant binding would only occur if the \gls{mab}/plastic
+  adhesion was quite fast and happened in the seconds prior to beginning
+  agitation. However, this problem is easily mitigated by agitating the tubes
+  with \gls{bsa} for several minutes prior to adding \gls{mab} to ensure even
+  mixing.
+\item[Improving protein detection --] While the \gls{bca} assay and \gls{elisa}
+  are quite precise, they both have problems that could lead to systemic bias as
+  well as increases in random noise. The \gls{bca} assay is non-specific. All
+  our data shows consistent small (\SI{0.5}{\ug}) but negative readings when
+  adding zero \gls{snb}, which indicates that some background protein (or
+  something that behaves like a protein) may be present that the \gls{bca} assay
+  is detecting. The \gls{elisa} is specific to \gls{mab}; however, in our case
+  we need to run a blank (just \gls{pbs}, \gls{bsa}, and \glspl{mab} without
+  carriers) and subtract this from the reading, effectively doubling the assay
+  variance. Using \gls{hplc} would mitigate both of these issues. \gls{hplc} can
+  specifically detect species based on differences in charge and size, so it
+  will likely be able to resolve \gls{stp} without the extraneous bias
+  introduced via the \gls{bca} assay. In the case of \gls{elisa} it will not
+  have remove the need to run a blank, but it likely will have lower variance
+  due to its automated nature.
+\end{description}
+
 \subsection{Additional Ligands and Signals on the DMSs}
 
 In this work we only explored the use of \acd{3} and \acd{28} \glspl{mab} coated