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