ADD section on reducing noise

This commit is contained in:
Nathan Dwarshuis 2021-09-02 17:34:42 -04:00
parent 7c427b39d8
commit edb3212b9b
1 changed files with 105 additions and 20 deletions

View File

@ -241,6 +241,7 @@
\newacronym{hepes}{HEPES}{4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid} \newacronym{hepes}{HEPES}{4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid}
\newacronym{nhs}{NHS}{N-hydroxysulfosuccinimide} \newacronym{nhs}{NHS}{N-hydroxysulfosuccinimide}
\newacronym{tocsy}{TOCSY}{total correlation spectroscopy} \newacronym{tocsy}{TOCSY}{total correlation spectroscopy}
\newacronym{hplc}{HPLC}{high-performance liquid chromatography}
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
% SI units for uber nerds % 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 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: 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 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} feature that is missing from the process currently is the use of \gls{gmp}
methods and materials. The microcarriers themselves are made from materials. The microcarriers themselves are made from porcine-derived collagen,
porcine-derived collagen, which itself is not \gls{gmp}-compliant due to its which itself is not \gls{gmp}-compliant due to its non-human animal origins.
non-human animal origins. However, using any other source of collagen should However, using any other source of collagen should work so long as the structure
work so long as the structure of the microcarriers remains relatively similar of the microcarriers remains relatively similar and it has lysine groups that
and it has lysine groups that can react with the \gls{snb} to attach \gls{stp} can react with the \gls{snb} to attach \gls{stp} and \glspl{mab}. Obviously
and \glspl{mab}. Obviously these would need to be tested and verified, but these these would need to be tested and verified, but these should not be
should not be insurmountable. Furthermore, the \gls{mab} binding step requires insurmountable. Furthermore, the \gls{mab} binding step requires \gls{bsa} to
\gls{bsa} to prevent adsorption to the non-polar polymer walls of the reaction prevent adsorption to the non-polar polymer walls of the reaction tubes. A human
tubes. A human carrier protein such as \gls{hsa} could be used in its place to carrier protein such as \gls{hsa} could be used in its place to eliminate the
eliminate the non-human animal origin material, but this could be much more non-human animal origin material, but this could be much more expensive.
expensive. Alternatively, the use of protein could be replaced altogether by a Alternatively, the use of protein could be replaced altogether by a non-ionic
non-ionic detergent such as Tween-20 or Tween-80, which are already used for detergent such as Tween-20 or Tween-80, which are already used for commercial
commercial \gls{mab} formulations for precisely this purpose\cite{Kerwin2008}. \gls{mab} formulations for precisely this purpose\cite{Kerwin2008}. Validating
Validating the process with Tween would be the best next step to eliminate the process with Tween would be the best next step to eliminate \gls{bsa} from
\gls{bsa} from the process. The \gls{stp} and \glspl{mab} in this work were the process. The \gls{stp} and \glspl{mab} in this work were not
not \gls{gmp}-grade; however, they are commonly used in clinical technology such \gls{gmp}-grade; however, they are commonly used in clinical technology such as
as dynabeads and thus the research-grade proteins used here could be easily 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 replaced. The \gls{snb} is a synthetic small molecule and thus does not have any
animal-origin concerns. 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. differences seen in comparison to the beads.
Experimentally, the first step involves separating the \glspl{dms} from the 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 (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{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 \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 good first pass experiment would be to analyze both populations with a T cell
differentiation/activation state flow panel first (since flow cytometry is differentiation/activation state flow panel first (since flow cytometry is
relatively cheap and doesn't require a large number of cells) to simply 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 need to be taken into account to find the bulk material properties of the
cross-linked gelatin\cite{Wang1984}. 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} \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 In this work we only explored the use of \acd{3} and \acd{28} \glspl{mab} coated