From edb3212b9b73fd555fadec0450117fb74398051e Mon Sep 17 00:00:00 2001 From: ndwarshuis Date: Thu, 2 Sep 2021 17:34:42 -0400 Subject: [PATCH] ADD section on reducing noise --- tex/thesis.tex | 125 +++++++++++++++++++++++++++++++++++++++++-------- 1 file changed, 105 insertions(+), 20 deletions(-) diff --git a/tex/thesis.tex b/tex/thesis.tex index e93aea4..47f5e53 100644 --- a/tex/thesis.tex +++ b/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