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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5943_Библиотеки_им_академика_М_И_Перельмана
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from gene therapy therapeutics is when two closely related proteins by amino acid
sequence exist, one endogenous and one coming from the gene therapy transgene.
For example, in the case of Factor IX protein, a modified gene and hence transgene
protein may have higher potency, but with only slightly different amino acid
sequence[13]. In this case, it may not be possible to use an immunoassay to distinguish between the wildtype and gene therapy transgene protein with an immunoassay; however, the two proteins will differ by mass, and so MS could quantify both
the wildtype endogenous and a transgene protein if that was of interest.
9.2.6.3 Semiquantitative Assay Formats
Both immunoassays and MS methods are quantitative. The ability to prepare
standards and quantitate unknown concentrations in biological matrices with total
error well under 40% is standard and typical for these two platforms. However,
immunoassay and MS platforms may not be sufficiently sensitive enough to detect
all types of transgenes. The LC–MS and immunoassays platforms described are
best suited for quantifying soluble proteins and so transmembrane proteins or
membraneassociated proteins and enzymes may not be able to be detected with
these platforms at sensitivities required. Transmembrane proteins or membrane
associated proteins and enzymes are not likely to be able to be detected without
extensive pretreatment of samples. For membranebound or associated transgene
proteins, it may be sufficient to use semiquantitative methodologies to assess
transgene protein biodistribution or relative expression levels.
Semiquantitative methods, techniques such as Western blotting and IHC are
typical formats used in gene therapy transgene protein expression determinations.
And the use of Western blots for quantification can nearly quantitative when
appropriate controls, standards are used[23]. In addition, as access to extensive
tissue samples is usually only available in Discovery or early preclinical studies,
Western blots for transgene protein quantification can be rapid to implement, easy
to carryout, and provide sufficient information to move programs toward clinical
development.
IHC is potentially the most useful technique to not only determine transgene
protein levels in various tissues but also provide information on transgene protein
levels within a cell[24]. Early discovery transgene protein biodistribution studies
may employ IHC as the primary technique for protein detection. Quantification
of transgene protein by computersupported image analysis can also be used;
however, standardization, normalization, and within laboratory comparisons
may be challenging[25]. Thus, IHC is useful and informative to show success of
gene therapy transgene protein expression, and although it may not be absolutely
quantitative, it can likely provide information to move a product forward to
clinical development.
ES ES EP[]
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9.3 Transgene Protein Activity Determination
Many investigational gene therapy programs are targeting genetic diseases and
aim to provide a normal copy of the gene to restore the function of the protein.
A variety of bioanalytical approaches to measure the expression in terms of
concentration have been described in earlier sections. However, when transgene
protein is an active enzyme or cofactor for other enzymes, the biochemical function of the protein in the form of protein activity measurement is more relevant
and the preferred approach. Enzyme activity assays measure the catalytic potential
of the enzyme by either measuring the consumption of substrate or production of
product over time with enzyme activity defined as moles of substrate converted
per time unit [26], for example, nmol/hour/mL of plasma or nmol/hour/mg of
protein. For coagulation factors such as factor VIII activity, the assay measures factor activity by either using a onestage clotting assay or a chromogenic substrate
assay. In this case, a unified international unit is used for activity measurement and
the factor concentration is expressed in international units per milliliter (IU/mL)
of human plasma, whereby 1IU/mL is equivalent to 100% factor activity[27].
Several factors, such as temperature, pH, enzyme or factor concentration, substrate concentration, and the presence of any inhibitors or activators, can impact
enzymatic reactions and the individual enzyme or cofactor dictates the assay conditions. This also leads to different measured activity values in different laboratories because of the slight differences in the assay conditions. From a bioanalytical
perspective, a functional protein activity assay is challenging to develop, validate,
and monitor as it can be impacted by many factors. This section will focus on
addressing protein activity method development challenges and providing guidance on method optimization, validation, and the use of the methods to support
sample testing. To limit the scope, the discussion will focus on using lysosomal
storage enzymes as an example; however, the presented information can be applied
to other protein activity assays, including protein activity for pharmacodynamic
biomarkers.
protein
9.3.1 Method Development Considerations
9.3.1.1 Enzyme Kinetics, theInitial Rate ofReaction, and
Substrate Concentration
Most enzyme reactions except for allosteric enzymes involving a single substrate
often follow Michaelis–Menten kinetics. The kinetics model takes the form of an
equation:
225
VS
KS
*[ ]
[]
Reaction velocity (V
)
Substrate concentration (S)
V
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Here, E is the enzyme, S is the substrate, ES is the enzyme–substrate complex,
and P is the product. The enzyme interacts with the substrate to form an enzyme–
substrate complex and leads to the generation of the product and the release of the
enzyme. The Michaelis–Menten equation as shown below shows how the initial
rate of reaction (V
V
0
) depends on the substrate concentration [S].
0
max
m
Here, V
for a given enzyme concentration. K
represents the maximum rate at saturating substrate concentration
max
is the Michaelis constant. At half V
m
max
, Km
is equal to the [S] Figure9.1[28].
Michaelis–Menten kinetics only applies to the initial velocity, therefore, the
reaction conditions must be chosen under the initial rate conditions such that the
activity is proportional to the concentration of enzyme over the entire dynamic
range. One underlying assumption is that substrate is not the limiting factor, and
the substrate concentration is sufficient to remain almost unchanged during the
reaction. During the initial method development stage, it is essential to perform
experiments to determine the initial rate of reaction and generate a substrate saturation curve to estimate the K
and V
m
. The initial rate window can be deter-
max
mined by generating a reaction progress curve via mixing the enzyme and its
substrate and measuring the time course of product generation. A given enzyme
may have a different reaction progress curve in different matrices and timecourse
experiments will need to be determined in the matrix of interest, Figure9.2[29].
A substrate saturation curve can then be generated by varying the substrate concentration in reactions occurring within the initial rate time window to determine
max
0
1/2V
max
K
m
Figure9.1 Michaelis–Menten Equation. “V0” denotes reaction velocity and “[S]” denotes
substrate concentration. V
enzyme concentration. K
reaction rate occurs.
is the highest reaction rate that can be attained for a given
max
is the concentration of substrate at which a half-maximal
m
6
80
Incubation time (h)
Activity (nmol/mL)
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226
60
40
20
0
012345
Figure9.2 Time course of an enzyme activity expressed in nmol/mL of the matrix.
Matrix 1 is represented by an open circle with a dashed line fitted through the linear
range of the reaction. Within 3 hours, the reaction is linear. Matrix 2 is represented by an
open square with a solid line fitted through the linear range of the reaction. Within
2 hours, the reaction is linear.
Matrix 1
Matrix 2
the substrate concentration for further method development. Ideally, it is best to
conduct an enzyme assay under substratesaturating conditions. However, some
substrates are quite costly like those for lysosomal storage enzymes, thus an alternative approach is to conduct the assay using a final concentration of K
as shown
m
in Ou etal. [30] to reduce overall cost. It is important to note that the velocity
should remain relatively stable throughout the whole incubation time when using
nonsaturating substrate concentrations.
9.3.1.2 Reference Standard
The source of reference standards for assay development is one of the biggest
challenges for protein activity assay development. The World Health Organization
(WHO) established biological reference materials (called International Standards)
as global reference standards for various antibodies, antigens, blood products and
related substances, cytokines, growth factors, endocrinological substances, and
invitro diagnostic devices [31]; however, WHO international standards are not
available for all biological materials such as lysosomal storage enzymes.
For lysosomal storage enzymes, 4Methylumbelliferone (4MU) derived artificial
substrates have been widely used for measuring lysosomal enzyme activity[4, 7].
The enzymatic reaction occurs at acidic pH 4.5–5.5 and the substrate is hydrolyzed by the enzyme to release 4MU, which is highly fluorogenic at pH 10–11.
Themeasured fluorescence intensity is proportional to the enzyme function. One
common reagent that is used as the reference standard for activity calculation is
the free fluorophore, 4MU [29, 32]. Since the reference standard (4MU)
227
4
5
Log fluorescence signal
2
Log fluorescence signal
(b)
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fluorescence is independent of the enzymatic reaction, for quantitative enzyme
activity measurement the use of recombinant enzyme is recommended during
method development and as additional controls to monitor the assay
performance.
During the early development phase when the assay is not wellcontrolled, a non
parallel relationship between enzyme activity and 4MU and a shift of x
intercept of
enzyme curves are often observed (Figure9.3a). The use of recombinant enzyme
can help to characterize the assay, to use as a tool for assay optimization to ensure
consistency and parallel relationship between enzyme reaction and 4MU standard
curve, and to help define the 4MU quantifiable range (Figure 9.3b). It is not
4
3
2
1
0
5
Enzyme
4MU
–4 –2 02
(slope = 0.4295)
(slope = 1.005)
Log concentration
(a)
4
3
2
1
0
Enzyme
4MU
–3 –2 –1 01
Log concentration
Figure9.3 Enzyme activity. Lysosomal storage enzyme activity in relation to 4MU for
activity calculation. Arecombinant enzyme can be used during method development to
optimize assay and to define the enzyme activity quantifiable range. (3A) Nonparallel
relationship between enzyme activity and 4MU. (3B) Parallel relationship between
enzyme activity and 4MU. The enzyme is represented by an open circle with a solid line
fitted through the enzyme curve. 4MU is represented by an open square with a solid line
fitted through the 4MU.
(slope = 1.009)
(slope = 0.995)
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Table9.1 Fluorescent background.
Vendor 1
(substrate
only)
Fluorescent
signal
It is necessary to evaluate the fluorescence signal of all reagents used in the enzymatic assay.
The table illustrates the fluorescent background of substrate 4Methylumbelliferyl α
galactopyranoside used in aGal A activity assay from Sigma (vendor 1), Cayman (vendor 2),
assay diluent, the minimum required dilution (MRD) 6 and MRD 10 plasma diluted in assay
diluent containing substrate from vendor 1.
2106 1462 389 1884 1952
Vendor 2
(substrate
only)
Assay
diluent
Human plasma at
MRD 6with
vendor 1 substrate
Human plasma at
MRD 10with
vendor 1 substrate
recommended to assign an activity unit per enzyme concentration and use the
recombinant enzyme as the reference standard for activity calculation as the production of commercially available enzymes is not well controlled and hence the
activity of each enzyme can vary from lot to lot. However, if assigning an activity
unit per enzyme lot helps to reduce assay variability, an extensive characterization
of a respective lot of enzymes should be implemented and a lottolot recombinant
enzyme bridging strategy should be established.
When generating the reference standard curve, it is essential to prepare the
standard curve containing similar reaction compositions as test samples as matrix
and substrate may have a different background than assay diluent. If matrix
effects are not an issue, standards can be prepared in buffered assay diluent.
However, it is necessary to include substrate in the standard curve preparation as
substrate tends to have a different fluorescence background as assay diluent and
background can also vary from vendor to vendor and lot to lot. Higher substrate
background may lead to an overestimation of the enzyme activity, Table 9.1.
Additional factors to consider for the standard curve are whether to institute background subtraction and the curve fit of choice. Background subtraction is
typically
applied. Linear and loglog linear fit are two commonly used curve fit models for
enzyme activity assay. Though the provided examples are specific to lysosomal
storage enzymes, the same principle applies to all quantitative enzyme assays
regarding the use of recombinant protein, curve fit, and ensuring the reference
standard contains similar reaction components as test samples.
9.3.1.3 Sample Processing
Enzyme activity assay can be measured in different types of matrices, for example,
plasma, serum, leukocyte, tissues, etc. Sample collection and sample processing are
important factors that can affect enzyme activity, but these factors are often
229
Donor 1 Donor 2 Donor 3 Donor 4
40
Enzyme activity (nmol/h/mL)
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overlooked during method development. The types of blood collection tubes and
downstream processing conditions such as centrifugation speed, storage
temperature,
and duration between collection to plasma/serum separation should be evaluated
early on to determine the most suitable and reproducible conditions[33]. The evaluation should include different donors and the reproducibility of the measured
enzyme activity should trump the conditions that give the highest measured enzyme
activity, Figure9.4. If the matrix for enzyme assay is leukocyte, it is important to
ensure laboratories are welltrained and qualified for leukocyte preparation.
Hemolysis can have an impact on downstream enzyme activity and protein concentration determination for leukocyte lysate. Additionally, the different means of transportation and shipping times should be evaluated to ensure good quality of leukocytes
can be generated at the processing laboratory.
During sample preparation for the activity assay measurement, the stability of
the enzyme under assay conditions should be evaluated and monitored. For
enzyme activity in leukocyte and tissue lysates, heat generation during the leukocyte sonication or tissue homogenization process may inactivate the enzyme. It is
important to evaluate the impact of temperature changes and derive a process to
best preserve the enzyme function.
30
20
10
0
ABCDE
Method
Figure9.4 Plasma sample processing comparison. Four donors (represented by different
filled patterns in the legend) with whole blood collected in K
different plasma preparation conditions. A: spin within 8 hours at 2400 revolutions per
minute (rpm) for 10minutes; B: spin within 8 hours at 3400rpm for 15minutes; C: spin
within 24 hours at 2400rpm for 10minutes; D: spin within 24 hours at 3400rpm for
15minutes. E: spin within 24 hours at 2465rpm for 20minutes. Each sample was
subjected to four independent measurements. Conditions B, D, and E were comparable
with better reproducibility. Conditions A and C yielded higher enzyme activity but poor
precision.
EDTA were subjected to
2
% Triton X-100
Enzyme activity (nmol/h/mL)
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230
9.3.1.4 Buffers and Incubation Temperature
Assay buffer is enzymedependent with slightly different compositions reported
in different literature for a given enzyme [29, 34]. During the method development, it is necessary to further optimize the buffer compositions and buffer pH
using the method described in the literature as a guide in the tested matrix.
Lysosomal storage enzymes are naturally found in the lysosomes, pH optimization is critical for this type of enzymatic assay. Additionally, some enzymatic
assays include the addition of surfactants, such as Tween20 and Triton X100, to
improve assay reproducibility. The impact of changes in surfactant percentage
should be evaluated as some assays are sensitive to small variations in surfactant
amount. For example, Triton X100may help to improve performance consistency
due to ease of pipetting, but the window of optimal Triton X100 percentage may be
very narrow, Figure9.5. Since the enzyme activity is measured by fluorescence signal
and correlated to 4MU for activity calculation, the preparation of assay buffer will be
critical as it can impact the overall calculated enzyme activity. In addition to buffer
composition and pH, the reaction temperature is also an important factor. Most
enzymatic assay occurs at body temperature (37°C). The impact of temperature
changes with respect to enzyme activity should also be evaluated during development.
9.3.1.5 Assay Dynamic Range, Minimum Required Dilution, Matrix
Interference, and Parallelism
Many gene therapy programs are targeting genetic diseases that lack a functioning
protein. These patients have little to no active protein present in their system.
Therefore, a quantitative and sensitive assay is likely needed to support drug
8
6
4
S1
2
0
Figure9.5 Impact of surfactants. Three donors represented by S1 (open circle), S2 (open
square), and S3 (open square) were tested in assay diluent containing different Triton-X %.
A significant shift in enzyme activity when the activity was measured in 0.05% vs. 0.1%
Triton X-100.
S2
S3
0.00 0.05 0.10 1.00
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development. In terms of assay dynamic range, published data can help to define
the initial target assay range and the required assay sensitivity during method
development. Some enzymes may require many folds over healthy donors’
range to achieve crosscorrection whereby the extracellular enzymes are taken up
by the enzymedeficient cells. An assay with a wider dynamic range will be helpful in this scenario.
Assay matrices are the trickiest component for assay development and careful
consideration of this variable in terms of the MRD, matrix interference, and parallelism should be evaluated during development. If matrix effects are not an
issue, standards can be prepared in buffered assay diluent like the majority of
biomarker assays instead of the matrix of test samples. To apply this approach, it
is crucial to demonstrate that the protein activity–response relationship in the
sample matrix is parallel to that of the buffered assay diluent through parallelism
evaluation. Parallelism experiments are performed through serial dilutions of a
highactivity sample in the buffered assay diluent using multiple individual
matrix lots (≥3lots during development). The consistency of the backcalculated
inrange concentrations in a dilution series not exceeding 30% coefficient of
variation (CV) or a relative bias approach by comparing serially diluted samples
to the first few dilutions to assess acceptable %Bias (20–30%) can be used to assess
parallelism. The latter is the preferred method. Additionally, parallelism experiments also help to define the MRD of the assay whereby the lowest dilution factor alleviates the matrix interference sufficiently and can achieve the required
assay sensitivity. In cases when parallelism cannot be performed due to a lack of
high protein activity samples, dilutional linearity should be tested in a similar
manner to parallelism.
For some protein activity assays, a matrix assay diluent is needed. As reported
in the literature, lysosomal sulfatases are strongly inhibited by sulfates, phosphates, and metal ions [29]. To reduce the inhibitory effects, one option is to
perform high sample dilutions to reduce the inhibitory effect, but this reduces the
assay sensitivity as well. An alternative approach is to use a heatinactivated
matrix with enzyme deactivated as the background matrix for the standard curve
and sample diluent. Though an inhibitory effect is present, the use of matrix assay
diluent will help to reduce the variability due to matrix interference. MRD can be
determined based on the targeted assay sensitivity and parallelism evaluation in
the matrix assay diluent.
normal
9.3.1.6 Specificity and Selectivity
The nonspecific activity of another enzyme or some matrix components can
result in a reduction or enhancement of the overall response, therefore, evaluation of specificity and selectivity are essential during assay development. The
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demonstration of specificity and selectivity should be evaluated in the context
of the assay as it can be difficult to demonstrate absolute specificity and
se
lectivity for enzyme activity assays. For example, the lysosomal enzyme
αNacetylgalactosaminidase has nonspecific activity toward the artificial
strate used for αgalactosidase A activity assay. To ensure speci-
sub
ficity, Nacetylgalactosamine (GalNAc) inhibitor is included in the
αgalactosidase A activity assay to eliminate any potential contribution from
Nacetylgalactosaminidase [35]. The optimal concentration of the inhibitor
should be determined during assay optimization to ensure the generated activity
is specific to the enzyme of interest. In some other cases, a second enzyme is
needed to cleave the intermediate substrates generated by the enzyme of interest,
like the two steps iduronidate2sulfatase enzymatic reaction. Understanding the
specificity of the enzymes involved in the reaction is needed. In this case, testing
the ability of the first enzyme to generate the intermediate substrate is an important factor. To perform enzyme specificity evaluations, heatinactivated plasma
with and without the addition of recombinant enzymes can be a viable approach.
As for selectivity, this parameter measures the ability of the assay to discriminate the analyte unequivocally in the presence of components that may be
expected to be present in the sample. To perform enzyme selectivity evaluations,
recombinant enzymes at a minimum double the endogenous activity can be used
to spike into individual donors (>5 donors during development) for this evaluation.
9.3.1.7 Quality Controls (QCs)
When a wellcharacterized enzyme or protein reference standard is available,
quality controls (QCs) generation is straightforward and can follow the approaches
used for the majority of biomarker assays. When a reference standard is based on
free fluorophore like 4MU, which is independent of the enzyme activity, the selection of the QC levels will be based on enzyme activity rather than the concentration of the enzyme. The use of recombinant enzymes can help to define the range
of quantitation and to set the upper limit of quantitation (ULOQ) and lower limit
of quantitation (LLOQ). It should be noted the raw responses of the recombinant
enzyme should fall within the raw response of the 4MU standard curve, Figure9.6.
Based on the enzyme activity of ULOQ and LLOQ, the remaining 3 QCs can be set
at about 75–85% of ULOQ for highquality control (HQC), at about 3 times the
LLOQ for lowquality control (LQC) and middlequality control (MQC) at mid
range of the standard curve. Sometimes it may be helpful to set two LLOQs and
finalize the selection during method validation. It may not be possible to obtain
donor samples with enzyme activity that can span either range of quantitation, a
combination of spiked QC with the recombinant enzyme in assay diluent and
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