CLARIOstar ® determines activity of a moss-produced human acid alpha- glucosidase (GAA) in a fluorescence-based assay.

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CLARIOstar® determines activity of a moss-produced human acid alpha-
glucosidase (GAA) in a fl uorescence-based assay
Enzymatic activity of moss-GAA - recombinantly expressed human alpha-glucosidase in moss P.patens
Microplate-based assay enables comparison to marketed alpha-glucosidase
CLARIOstar
®
supports assay optimization and quick enzyme quantifi cation in samples from moss cultivation
Birgit Berg1, Anita Fischer1, Fabian Hässler1, Paulina Dabrowska-Schlepp1, Franka Maurer2 and Stefanie Tintelnot2
1Greenovation Biotech GmbH, Germany, 2BMG LABTECH GmbH, Ortenberg, Germany
Introduction
The lysosomal enzyme acid alpha-glucosidase
(GAA, Uniprot: P10253) is a hydrolase responsible
for degradation of lysosomal glycogen to glucose.
Genetically inherited defi ciency of the GAA is known as
Pompe disease. Patients suffering from this disorder
accumulate glycogen in various body tissues – especially
in skeletal and respiratory muscles. Currently, Myozyme®
and Lumizyme® (Sanofi Genzyme) - recombinant forms
of human alfa glucosidase produced in Chinese Hamster
Ovary (CHO) cells are the only approved treatments for
Pompe disease.
Moss-GAA is an alternative recombinant version of
human GAA produced in moss Physcomitrella patens.
This proprietary expression system profi ts from
advantages, such as: eliminated risk of contamination
by zoonotic pathogens and easy manipulation of the
glycosylation pathway. Additionally, moss-made proteins
exhibit highly homogenous glycosylation profi le, which
can be benefi cial for targeted uptake of lysosomal
enzymes, such as GAA.
The GAA producing moss strains are cultured in
illuminated bioreactors and secrete moss-GAA into the
cultivation supernatant. To measure the activity and
quantify the recombinantly produced enzyme in culture
samples, we established a known fl uorescence-based
activity assay in a 96-well plate format, optimized its
performance and conducted assay qualifi cation and
initial validation.
Materials & Methods
CLARIOstar
®
microplate reader (BMG LABTECH)
4-methylumbelliferyl α D-glucopyranoside (Roth)
CHO-GAA–Myozyme
®
(Sanofi Genzyme)
Bovine Serum Albumin (Sigma)
Black fl at-bottomed 96-well microplates (Roth)
Shaker Titramax 1000 (Heidolph)
Incubator (Binder)
Experimental Procedure
20 µL of GAA containing sample was incubated with 80 µL
assay buffer (56 mM citric acid, 88 mM Na2HPO4, 0,4% BSA,
pH 4.4) containing 0.25 mM 4-MU α -D-glucopyranoside in
a black 96-well microplate. The plate was agitated for 10
sec. at 900 rpm, covered with an adhesive microplate seal
and incubated at 37°C protected from light for 60 min. The
reaction was stopped by adding 200 µL of stop solution (0.1
M Glycine, 0.1 M NaOH) and fl uorescence was measured
as outlined below.
Rev. 09/2019
Keywords: 4-Methylumbelliferone; Moss; Glucosidase, Enzyme activity
Assay principle
Fluorescence-based enzyme activity assay using
4-methylumbelliferyl-α-D-glucopyranosid has been ex-
tensively used for the measurements of GAA
concentration and activity in different types of samples.
In this assay GAA hydrolyses the substrate to release
the 4-methylumbelliferone that can be measured
fl uorometrically. We used CHO-GAA-Myozyme
®
as a
standard for assay development, optimization and its
qualifi cation. Subsequently the developed assay was
adapted (examination of possible matrix effects) for use
with culture media samples originating from the moss
cultivation.
Optic
settings
Fluorescence intensity, endpoint
Monochromator
Excitation 360-20
Dichroic: auto 402,5
Emission 450-30
General
settings Number of
fl ashes
40 per well
Settling time 0.2 s
Instrument settings
O
O
CH
3
O
O
OH
HO
HO
OH
OH
O
OH
HO
HO
OH
O
CH3
HO O
+ H2O+
alpha-glucosidase
4-Methylumbelliferyl alpha-D-glycopyronoside
alpha-D-glucose
4-Methylumbelliferon
Results & Discussion
1. Linearity
Calibration curve fi tting is a critical component of assay
performance. We assessed linear regression and 4PL
function for quality of the curve fi t by comparison of
recovery rates for known amounts of CHO-GAA within
the range 39,06 – 4000 ng/mL. Signifi cantly better fi t of
the calibration curve, based on the visual assessment
as well as better recovery rates between 100% –
120% throughout the whole working range have
been achieved with 4-PL fi t, which was chosen as the
assay’s calibration function.
Fig. 1: Assay principle for the measurement of enzymatic activity of
GAA
NEPHELOMETRY AlphaScreen®FP TRF & TR-FRET LUM & BRET
AN
339
FI & FRET ABSORBANCE

Conclusion
This application note describes the adaptation,
optimization and qualifi cation of a fl uorometric enzyme
activity assay for acid alpha-glucosidase in 96 well plate.
Microplate-based format enables fast and simultaneous
quantifi cation of moss-GAA concentrations in multiple in-
process samples. Analyzed assay parameters: linearity,
sensitivity and precision demonstrate satisfactory ranges
for the intended assay use.
The fi lter-based microplate reader CLARIOstar
®
from
BMG LABTECH provides an easy-to-use instrument, which
measures this fl uorometric assay quickly and accurately.
Furthermore, the complementary MARS Analysis Software
Module is extremely helpful for evaluating and analyzing
assay results.
4. Specifi c enzyme activity
The described assay was subsequently used to measure
specifi c enzyme activity of different GAA prepa-
rations. For this an appropriate standard curve with
4-methylumbelliferone has been included in the micro-
plate layout. The specifi c activity of recombinant GAA
versions: CHO-GAA and moss-GAA has been measured,
showing comparable values for both enzymes.
Specifi c activity (µmol/mg/min)
rhGAA Mean SD CV%
CHO-GAA 9,9 1,5 15,1
Moss-GAA 9,8 1,4 13,9
2. Sensitivity (LOQ)
Limit of quantitation (LOQ) has been assessed based
on the CV% of the CHO-GAA spike throughout the
concentration range. The CV% increases above the 10%
between the 31 and 39 ng/mL. This result corresponds
well to the value based on customary signal -to-noise ratio
1:10 (interpolated from the mean absorbance of the blank
+ 10xSD of the blank), which was calculated to 36 ng/mL.
The LOQ of the assay was set to 36 ng/mL.
3. Precision
Intra-assay precision was assessed by analysing three
CHO-GAA samples in respective dilutions (2 dilutions
per sample, each dilution in triplicate) covering the
relevant working range. Intra-assay precision of %CV ≤ 5%
was found. For the estimation of inter-assay precision
corresponding samples were measured in following
experiment, performed on a different day, by another
operator. Inter-assay precision of %CV ≤ 8% was
determined.
Table 2: Comparison of specifi c enzyme activity between
the marketed version of GAA and the moss product (n=4)
Intra-assay precision
CHO-GAA
[ng/mL]
Dil.
factor Mean SD CV%
5000 10 4734,79 175,54 4%
5
2500 10 2354,01 31,32 1%
5
1250 10 1267,94 59,38 5%
5
Inter-assay precision
CHO-GAA
[ng/mL]
Dil.
factor Mean SD CV%
5000 10 5045,29 359,80 7%
5
2500 10 2501,84 157,43 6%
5
1250 10 1362,98 1135,51 8%
5
A
B
Myozyme concentration ng/mL
0 500 1000 1500 2000 2500 3000 3500 4000 4500
250000
200000
150000
100000
50000
0
Myozyme concentration ng/mL
RFU
0 500 1000 1500 2000 2500 3000 3500 4000 4500
250000
200000
150000
100000
50000
0
EC50 = 8335,02
Slope = 1,02
Top = 691297,5
Bottom = 518,11
R2 = 0,9999
y = 56,14 * x+5505
R2 = 0,9893
RFU
Fig. 2: Assessment of the calibration curve fi t: A) linear regression;
B) 4-PL. Red markers represent spike recovery values (n=4).
Black markers represent data used for generation of the
calibration curve (n=2).
Table 1: Precision of GAA assay
CLARIOstar® Plus
PHERAstar®FSX Omega series