Haemophilia (2014), 20, 593–600
12374
ORIGINAL ARTICLE Laboratory science
Evaluation of the activated partial thromboplastin time
assay for clinical monitoring of PEGylated recombinant
factor VIII (BAY 94-9027) for haemophilia A
J-M. GU,* P. RAMSEY,* V. EVANS,* L. TANG,† H. APELER,† L. LEONG,* J. E. MURPHY,†
V . L A U X * and T . M Y L E S *
*Hematology Research; and †Biological Research, US Innovation Center, Bayer HealthCare Pharmaceuticals, San Francisco,
CA, USA
Summary. Patients with haemophilia (PWH) are
usually monitored by the one-stage activated partial
thromboplastin time (aPTT) factor VIII (FVIII) assay.
Different aPTT activators may affect clotting time
(CT) and FVIII:C levels in patients treated with
PEGylated FVIII. To evaluate the characteristics of
PEGylated FVIII (BAY 94-9027) in various aPTT
clotting assays, and to identify suitable aPTT reagents
for monitoring BAY 94-9027 during the treatment of
PWH, BAY 94-9027 and World Health Organization
(WHO) 8th FVIII standards (WHO-8) were spiked
into pooled and individual severe haemophilia A
plasma at 1.0, 0.25 and 0.05 IU mL 1. Five
commercial aPTT reagents widely used in clinical
laboratories were compared and evaluated for BAY
94-9027 activity in plasma from PWH. BAY 94-9027
and WHO-8 bestowed similar CT and excellent
precision when ellagic acid (SynthAFax, Dade Actin,
and Cephascreen) aPTT reagents were used. In
Introduction
Prophylactic treatment with recombinant or plasma-
derived factor VIII (FVIII) is preferred in children with
severe haemophilia to prevent bleeding and joint damage
[1,2]. A long-acting FVIII would substantially improve
treatment options for patients with haemophilia A by
reducing the number of injections, especially for infants
and patients with problematic venous access [2,3].
Attachment of polyethylene glycol (PEG) has been
considered an effective method of prolonging the half-
life of recombinant therapeutic proteins [4–11].
Correspondence: Jian-Ming Gu, Bayer HealthCare, LLC, 455
Mission Bay Blvd South, San Francisco, CA 94158, USA.
Tel.: +1 415 437 5901; fax: +1 415 437 5968;
e-mail: jian-ming.gu@bayer.com
Accepted after revision 18 December 2013
© 2014 John Wiley & Sons Ltd
DOI: 10.1111/hae.
contrast, BAY 94-9027 showed significantly
prolonged CT and poor precision compared with
WHO-8 using silica aPTT reagents (APTT-SP and
STA PTT 5). Furthermore, free 60-kDa polyethylene
glycol (PEG), used for the conjugation of FVIII,
showed a dose-dependent prolongation of CT in the
APTT-SP assay. There was no effect on the
SynthAFax-APTT, prothrombin time, or FXIa-
initiated thrombin generation assay, demonstrating
that the PEG moiety on FVIII has no general effect on
the coagulation cascade. In summary, ellagic aPTT
reagents (SynthAFax, Dade Actin, and Cephascreen)
are most suitable for evaluating potency of BAY 94-
9027 and should be the preferred aPTT reagents used
in clinical laboratories for monitoring FVIII activity
after infusion of BAY 94-9027 to PWH.
Keywords: aPTT assays, clotting time, coagulation tests,
factor VIII, haemophilia, PEGylation
BAY 94-9027 is a site-specific PEGylated FVIII in
which a 60-kDa PEG is conjugated to a cysteine resi-
due on the light chain of B-domain–deleted FVIII.
BAY 94-9027 is fully active in vitro and in vivo, with
prolonged pharmacokinetics in preclinical species
[5,12], and showed significantly less immunogenicity
in haemophilia A mice, normal rats and rabbits com-
pared with non-PEGylated recombinant FVIII (rFVIII)
[13]. BAY 94-9027 is well tolerated in preclinical
experiments [5], and showed improved pharmacoki-
netics in clinical phase I studies [14]. A phase II/III
clinical study with BAY 94-9027 is ongoing for
prophylactic therapy of haemophilia A.
The activated partial thromboplastin time (aPTT)
assay plays an essential role in clinical management of
patients with haemophilia [15,16]. Precise measurement
of FVIII activity is essential for monitoring on-demand
and regular prophylactic therapy. However, it is well
593
594 J-M. GU et al.
recognized that there may be considerable inter- and
intra-laboratory variation for the one-stage clotting
assay due to differences in the types of clotting activa-
tors and sources of phospholipids (PLs) used in the com-
mercially available aPTT reagents, and differences in the
instrumentation platforms used [17–24]. The chromo-
genic assay is more precise for determining FVIII activity
than the one-stage clotting assay but has limited usage
in the clinic [17]. There is a need to identify reliable
aPTT reagents that are compatible with the new
generation of therapeutics for treating haemophilia.
The objective of this study was to evaluate different
clinical aPTT clotting assays and the extent of
variability between reagents, to provide extensive
evaluation of the characteristics of PEGylated FVIII
(BAY 94-9027) in those assays, and to identify
suitable clinical assays for monitoring BAY 94-9027
during treatment of patients with haemophilia A.
Materials and methods
Study materials
A pool of severe haemophilia A plasma (HRF Inc.,
Raleigh, NC, USA) from three donors and plasma from
five individual donors (without FVIII inhibitors and
FVIII:C <1%) were used. Normal-control assayed
plasma was from Instrumentation Laboratory (Bedford,
MA, USA). The WHO 8th International Standard for
blood coagulation FVIII:C (WHO-8, lot#07/350) was
from the National Institute for Biological Standards
and Control (NIBSC, UK) [25]. BAY 94-9027, an
investigational PEGylated rFVIII drug product
(lot#949027T070209), was provided by Bayer
HealthCare (Berkeley, CA, USA). The WHO-8 and
BAY 94-9027 were spiked into pooled severe haemo-
philia A plasma to reach FVIII levels of 0.05 IU mL 1
(low), 0.25 IU mL 1 (medium), and 1.0 IU mL 1
(high). Equivalent spiking of BAY 94-9027 into five
individual plasma donors with severe haemophilia A
was performed to test donor variability. Spiked plasma
was aliquoted and stored at 80°C until analysis.
Reagents, coagulation analysers and test protocols
The aPTT reagents, including the type of activators,
PLs, vendors and coagulation analysers used in this
study are summarized in Table 1.
Table 1. aPTT reagents used.
Reagents Activator
SynthAFax Ellagic acid
APTT-SP Colloidal silica
Dade Actin Ellagic acid
Cephascreen Polyphenol (ellagic acid)
STA PTT 5 Particulate silica
IL, Instrumentation Laboratory; PL, phospholipid; aPTT, activated partial thromboplastin time.
Haemophilia (2014), 20, 593--600
ACL-TOP analyser. The one-stage clotting assay was
performed on an automatic ACL-TOP coagulometer
(Instrumentation Laboratory). An optical (671 nm)
read-based clot detection was used to minimize inter-
ference from lipemia, haemoglobin and bilirubin. Two
aPTT reagents [APTT-SP: synthetic PLs and silica (He-
mosILâ), APTT-SF: SynthAFax (SF), Instrumentation
Laboratory] were used. The calcium reagent was
0.025 M for SP and 0.02 M for the SF-based assay.
The testing protocol for SynthAFax in ACL TOP was
based on the ACL-TOP test parameters for the APTT-
SP reagent. Briefly, the existing test protocol was
copied to create an APTT-SF protocol with a 3-min
incubation according to the manufacturer recommen-
dations. Data acquisition was set at 120 s, and data
reduction and mathematic algorithms to determine the
clotting endpoint were the same.
STAGO STA analyser. The one-stage clotting assay
was also performed on the STA Compact Analyser
(Diagnostica Stago, Parsippany, NJ, USA), which
detects clot formation electromagnetically. Three
brands of aPTT reagents STA PTT 5, Stago Cepha-
screen, and Dade Actin Activated Cephaloplastin
reagent (Siemens, Marburg, Germany) were used on
the STA Compact with three user-defined test setups
created based on the manufacturer’s default aPTT
assay. A quantity of 50 lL of plasma sample was
added to 50 lL of aPTT reagent. The mixture was
incubated at 37°C for 240 s, after which 50 lL of
0.025 M calcium chloride was added and measurement
of clot time (CT) was initiated. For determination of
FVIII potency, pooled plasma from patients with
severe haemophilia A (<1% FVIII activity) was spiked
with a WHO-8 calibrator and the resulting CTs
plotted using third-party statistical software.
FVIII activity by aPTT assay. For FVIII activity mea-
surements, WHO-8 [25] was used as the reference cal-
ibrator. The reference material was reconstituted with
1 mL of distilled water according to manufacturer
instructions, and then diluted to a 1.0 IU mL 1 con-
centration of FVIII with pooled severe haemophilic
plasma. The one-stage clotting assay was performed
using the following reagents from Instrumentation
Laboratory: WHO standard (150%, 100%, 50%,
25%, 10%, 5%, 2% and 0% automatically diluted
1:10 in Factor Diluent solution with each dilution
Phospholipid Vendor Analyser
Synthetic PL IL ACL TOP
Synthetic PL IL ACL TOP
Cephalin from rabbit brain Siemens STAGO STA Compact
Cephalin from rabbit cerebral tissue STAGO STAGO STA Compact
Cephalin from rabbit cerebral tissue STAGO STAGO STA Compact
© 2014 John Wiley & Sons Ltd

tested in duplicate), pooled FVIII-deficient plasma;
APTT-SP or SynthAFax and 0.025 M calcium chloride
solution. Samples were tested in triplicate to reduce
the influence of assay variability. Samples with FVIII
activities <10% were remeasured using a different
dilution of plasma and factor diluent on the same cali-
bration curve.
FVIII activity by chromogenic assay. FVIII activity
was measured by the two-stage chromogenic assay
(Biophen FVIII:C; Aniara, West Chester, OH, USA)
according to the manufacturer instructions, except
that the reference standards were calibrated against
WHO-8. In the first stage, FVIII was activated by
thrombin to FVIIIa (which promotes conversion of FX
to activated FXa in the presence of FIXa), PLs, and
CaCl2 in Tris buffer supplied by the kit. In the second
stage, a specific FXa chromogenic substrate (SXa-11)
was added, and absorbance at 405 nm was deter-
mined using a SpectraMax M5 microplate reader
(Molecular Devices, Sunnyvale, CA, USA). Activity of
FXa is proportional to the FVIII activity of the sam-
ple. Each dilution of the calibrator and samples was
tested in triplicate.
Thrombin generation assay with FXIa. A thrombin
generation assay (TGA) in platelet-poor plasma using
the Thrombinoscope BV and Thrombinoscope BV
reagents (Stago, Maastricht, the Netherlands) was per-
formed as described by the manufacturer [26,27] with
minor modifications. Briefly, pooled severe haemophil-
ic plasma (HRF Inc.) was thawed at 37°C and spiked
with BAY 94-9027 or sucrose-formulated rFVIII
(rFVIII-FS) at a final concentration of 1 IU mL 1. A
quantity of 20 lL of the mixture of human FXIa (final
concentration, 10 pM) and microparticles containing
PL was added to 80 lL of plasma and equilibrated at
37°C for 10 min. The reaction was initiated by adding
20 lL of calcium and thrombin substrate solution
(Flu-substrate). Human FXIa (Haematologic Technol-
ogies, Inc., Essex Junction, VT, USA) was used to
activate thrombin generation. The fluorescence
Table 2. Clotting time (CT) and precision (CV) of two different reagents analysed on ACL-TOP analyser.
Tests n CT, s
APTT-SF
Within run 20
Between run 60
APTT-SP
Within run 20
Between run 60
APTT, activated partial thromboplastin time; SF, SynthAFax; SP, silica; CT, clotting time; CV, coefficient of variation.
*F8DP (FVIII-deficient plasma) was spiked with 1 IU mL 1 BAY 94-9027.
†
P < 0.001 compared to normal-control plasma.
‡
Out of accepted manufacturer limits for ACL-TOP analyser (<2.5%).
© 2014 John Wiley & Sons Ltd
EVALUATION OF APTT ASSAYS FOR BAY 94-9027 595
development was continuously monitored in real time
for 60 min using an excitation filter at 390 nm and an
emission filter at 460 nm. Thrombin generation
parameters derived from the thrombin generation
curve include lag time (time to 16.7% of peak concen-
tration; minutes), endogenous thrombin potential
(ETP; area under the curve; nM minutes), peak height
thrombin (nM), time to peak (minutes) and time to
tail (minutes).
Statistical analyses
Dunn post statistic tests were performed with Graph-
Pad Prism 5 (GraphPad Software Inc, La Jolla, CA,
USA). A two-tailed P value <0.05 was considered sta-
tistically significant. Correlation coefficients (r) were
calculated according to Spearman’s test in Prism 5.
The data were expressed as mean values with SD and
coefficient of variation (%CV). All summaries and sta-
tistical analyses are based on actual values. Between
run precision was calculated from values obtained on
each of the three assay days.
Results
Within-run and between-run precision on the ACL
TOP: effect of activators on aPTT assay
BAY 94-9027 was spiked into pooled FVIII-deficient
plasma to evaluate within-run and between-run preci-
sion of the method and the ACL-TOP instrument.
With SynthAFax (APTT-SF) as an activator, mean CT
of 1 IU mL 1 BAY 94-9027 in FVIII-deficient plasma
was similar to that of normal-control assayed plasma
(23.0  0.4 vs. 22.8  0.6 s respectively; n = 20,
P > 0.05; Table 2). The within-run %CV for BAY 94-
9027 and normal-control plasma was similar and
acceptable for a coagulation-based clinical assay
(1.9% vs. 1.7%). BAY 94-9027 spiked samples
(1 IU mL 1) were prepared on three separate days,
and between-run %CV was 2.5% for BAY 94-9027,
which was similar to normal control and considered
BAY 94-9027 in F8DP* Normal-control assayed plasma
Mean  SD Mean  SD
CV, % CT, s CV, %
23.0  0.4 1.9 22.7  0.4 1.7
22.8  0.6 2.5 22.6  0.4 1.7
45.5  3.8† 8.3‡ 29.6  0.5 1.7
45.4  3.9† 8.6‡ 29.8  0.5 1.8
Haemophilia (2014), 20, 593--600
596 J-M. GU et al.

acceptable for a clinical assay. However, using the sil-
ica activator (APTT-SP), mean aPTT clotting time of
BAY 94-9027 was significantly prolonged compared
with normal-control assayed plasma (45.5  3.8 vs.
29.6  0.5 s respectively; n = 20, P < 0.001). The
within-run %CV of BAY 94-9027 was also signifi-
cantly higher than that of normal control (8.3%
vs. 1.7%). Similar mean APTT-SP clotting times and
%CV were also obtained for BAY 94-9027 in a
between-run comparison. These data clearly indicate
that ellagic acid-based aPTT reagents (e.g. APTT-SF)
are suitable and reliable for evaluating potency of
BAY 94-9027 in plasma.
Method comparison study. Five aPTT reagents were
evaluated on two different instrument platforms for
BAY 94-9027 and WHO-8 FVIII concentrates; a sum-
mary of the mean CTs and %CV of three replicate
determinations at each concentration are shown in
Table 3.
For the ACL-TOP analyser, mean aPTT clotting
time of BAY 94-9027 was very similar to that of
WHO-8–spiked pooled severe haemophilic plasma
when activated by SynthAFax (%CV varied between 0
and 2.1). Mean CT of individual plasma spiked with
BAY 94-9027 was similar to that of pooled plasma
for BAY 94-9027; although variability from individual
plasma appears slightly higher (%CV, 7.4–8.4%), it is
still within an acceptable range. However, significantly
prolonged CT and higher %CV (range, 3.0–17.0%)
for BAY 94-9027 at all three concentrations were
obtained in the silica-based aPTT assay (APTT-SP)
compared with WHO-8–spiked plasma.
With the STAGO STA analyser, similar mean CTs
and good precision were found for both BAY 94-9027
and WHO-8 when Dade Actin and Cephascreen were
used as activators. However, the CTs for BAY
94-9027 were significantly prolonged in the silica-
based aPTT reagent (STA PTT 5), similar to experi-
ments performed on the ACL TOP. As summarized in
Table 3, significantly prolonged CT and high vari-
ability were observed (%CV: 1.4–17.4%) for BAY
94-9027 at all three concentrations when STA PTT 5
was used as the activator.
Method comparison studies in BAY 94-9027-spiked
haemophilia A plasma samples demonstrated high
agreement among ACL TOP/SynthAFax, STAGO/
Dade Actin, and STAGO/Cephascreen (r = 0.98 and
0.95, Fig. 1a,b). Among all five reagents tested, the
ellagic acid-based aPTT reagents (SynthAFax, Dade
Actin) and the polyphenol reagent (Cephascreen)
showed the best correlation, even between the two dif-
ferent instruments (Fig. 1a). Conversely, we observed
a considerable disparity between ACL TOP/APTT-SP
and STAGO/PTT5, which are all silica-based reagents
(r = 0.88, Fig. 1c). As expected, there is very poor
correlation between APTT-SP and SynthAFax using
the ACL-TOP analyser (r = 0.84, Fig. 1d).
These data demonstrate that the silica-based aPTT
assay is an unreliable assay for BAY 94-9027, and the
use of a WHO-8 calibrator with this assay would
markedly underestimate FVIII levels in patients treated

Table 3. Summary of the mean clotting time (CT) and precision (CV) using different aPTT reagents and analysers.

WHO-8 BAY 94-9027

Pooled plasma (n = 3) Pooled plasma (n = 3) Individual plasma (n = 5)

Mean  SD Mean  SD
aPTT reagent FVIII:C (IU mL 1) CT, s CV, % CT, s CV, % Mean  SD CT, s CV, %
ACL TOP SynthAFax 1.0 23.7  0.2 0.6 23.3  0.3 1.3 24.2  1.8 7.4
0.25 30.3  0.3 1.1 28.6  0.6 2.1 29.6  2.3 7.8
0.05 36.7  0.4 1.0 36.0  0.2 0 37.1  3.1 8.4
<0.01 49.3  0.3 0.6 49.3  0.3 0.6 50.3  3.7 7.4
APTT-SP 1.0 32.2  0.2 0.7 47.7  2.1* 4.4 52.9  9.0* 17.0
0.25 40.4  0.2 0.5 60.7  3.3* 5.4 64.4  7.9* 12.3
0.05 50.4  0.3 0.5 74.6  5.8* 7.8 74.2  2.2* 3.0
<0.01 101.5  0.5 0.6 101.5  0.5 0.6 99.3  3.8 3.8
STAGO STA Dade Actin 1.0 32.0  0.1 0.3 30.1  0.1 0.3 30.5  3.4 7.4
0.25 39.7  0.3 0.8 36.9  0.6 1.6 37.9  4.2 11.1
0.05 50.2  0.0 0.0 46.8  0.5 1.1 47.9  5.0 11.0
<0.01 72.9  0.1 0.1 72.9  0.1 0.1 74.2  0.4 0.1
Cephascreen 1.0 33.6  0.3 0.9 32.5  0.0 0.0 34.0  3.4 9.9
0.25 41.8  0.0 0.0 40.5  0.1 0.2 42.0  4.2 10.0
0.05 52.9  0.1 0.1 51.7  0.5 1.0 53.6  5.7 10.6
<0.01 79.0  0.4 0.5 79.0  0.4 0.5 78.2  0.7 0.9
STA PTT 5 1.0 36.8  0.1 0.3 46.6  1.3* 2.8 50.1  8.7* 17.4
0.25 46.3  0.0 0.0 56.9  0.8* 1.4 60.6  9.2* 15.2
0.05 59.2  0.2 0.1 72.7  2.3* 3.2 78.4  11.5* 14.7
<0.01 115.4  1.1 1.0 115.4  1.1 1.0 115.4  1.1 1.0
aPTT, activated partial thromboplastin time; FVIII, factor VIII; CV, coefficient of variation; WHO-8, World Health Organization 8th International
Standard FVIII concentrate.
*P < 0.001 compared with WHO-8.

Haemophilia (2014), 20, 593--600 © 2014 John Wiley & Sons Ltd
 EVALUATION OF APTT ASSAYS FOR BAY 94-9027 597

(a) (b)

(c) (d)

Fig. 1. Correlation between activated partial

thromboplastin time (aPTT) values obtained using
different aPTT reagents on ACL-TOP and STAGO
analysers: (a) SynthAFax vs. Dade Actin, (b) Ceph-
ascreen vs. Dade Actin, (c) APTT-SF vs. STA-
PTT5, and (d) APTT-SP vs. SynthAFax.

with BAY 94-9027. Indeed, only ~10% recovery was
obtained for BAY 94-9027 with FVIII:C measurement
when WHO-8 was used as the calibrator and APTT-
SP as the activator (data not shown). In contrast,
FVIII:C mean levels and precisions (%CV) of WHO-8
and BAY 94-9027 in the spiked plasma measured by
ellagic acid-based aPTT assay (e.g. Cephascreen) are
comparable to those measured by chromogenic assay
at the concentrations tested (1.0, 0.25 and
0.05 IU mL 1; Table 4).
Clotting wave analysis. The ACL-TOP analyser soft-
ware allows display of the clot-reaction curves and
also displays the associated first- and second-derivative
curves. These derivative plots are calculated by the
ACL-TOP software from absorbance data (671 nm)
and reflect the velocity (first-derivative) and accelera-
tion (second-derivative) at various points throughout
the clotting reaction, which is depicted as the clot-
reaction curve. The inflection point of the second-
derivative point (peak acceleration point) defines the
clotting endpoint. The clotting endpoint for all
reagents was taken as the second-derivative accelera-
tion peak. We compared second-derivative curve
patterns in a range of BAY 94-9027– and WHO-8–
spiked plasma tested using two aPTT reagents.
The aPTT assay utilizing the ellagic acid activator
(SynthAFax) shows that both BAY 94-9027 and
WHO-8 have similar second-derivative curve patterns
at each concentration; the peak and patterns are

Table 4. FVIII:C activity and precisions (%CV) of WHO-8 and BAY 94-9027 analysed by chromogenic assay and aPTT assay.
1
Assay FVIII, IU mL 1.00 0.25 0.05
WHO-8 Chromogenic Mean  SD 1.03  0.07 0.22  0.02 0.04  0.01
CV (%) 6.71 9.4 25.0
aPTT Mean  SD 1.00  0.05 0.26  0.00 0.04  0.00
CV (%) 4.68 0.00 1.12
Chromo/aPTT ratio 1.03 0.85 1.0
BAY 94-9027 Chromogenic Mean  SD 1.10  0.07 0.25  0.02 0.04  0.01
CV (%) 6.30 6.05 13.51
aPTT Mean  SD 1.12  0.00 0.32  0.01 0.05  0.01
CV (%) 0.00 1.15 7.83
Chromo/aPTT ratio 0.98 0.78 0.80
Chromogenic activity measured by Hyphen FVIII:C kit; One-stage APTT measured by STAGO Cephascreen.
aPTT, activated partial thromboplastin time; CV, coefficient of variation; FVIII, factor VIII; WHO-8, World Health Organization 8th International
Standard FVIII concentrate.

© 2014 John Wiley & Sons Ltd Haemophilia (2014), 20, 593--600
598 J-M. GU et al.
(a) (b)
(c) (d)
dependent on the FVIII concentrate tested (Fig. 2a and
b). A single acceleration peak was seen in high FVIII
plasma samples (1 IU mL 1). However, atypical sec-
ond-derivative curves with the appearance of extra
shoulder peaks or double peaks were seen in low
FVIII samples (0.25 and 0.05 IU mL 1). The CT was
usually given on the initial peak. It appears that with
lower FVIII levels, a lower initial second-derivative
acceleration peak is observed.
In the aPTT assay with silica reagent (APTT-SP),
similar second-derivative patterns to the SF activator
were seen in WHO-8–spiked haemophilia A plasma
(Fig. 2c). However, BAY 94-9027 with equivalent
spiked FVIII levels displayed much lower initial sec-
ond-derivative acceleration peaks and also significantly
delayed appearance of the initial peak, as reflected in
prolonged CTs compared with the SF activator
(Fig. 2d).
Effect of PEG on contact activation by silica matrices
in aPTT assay. BAY 94-9027 displays significantly
delayed CT and altered wave form signatures in the
silica-based aPTT assay, possibly due to interference
of contact activation on the silica surface by the 60-
kDa PEG moiety of the conjugated FVIII. To test this
possibility, APTT-SF (ellagic acid), APTT-SP (silica)
and prothrombin time (PT; RecombiPlasTin 2G, He-
mosIL, Instrumentation Laboratory) assays were per-
formed in the FVIII-deficient plasma spiked with
rFVIII-FS and BAY 94-9027 at 1 IU mL 1, and the
effect of free PEG on prolongation of CT was deter-
mined. Free 60-kDa PEG at 0, 1, 3, 10, 30 and
100 lg mL 1 were used in these assays. Similar to
BAY 94-9027, free PEG had no effect on the CT in
haemophilia A plasma spiked with rFVIII-FS using the
APTT-SF and PT assays. However, free PEG dose-
dependently prolonged CT in the APTT-SP assay, and
PEGylated FVIII at 1 IU mL 1 had a similar effect on
Haemophilia (2014), 20, 593--600
Fig. 2. Clotting wave curve analyses tested using
(a and b) APTT-SF or (c and d) APTT-SP assays.
Black, red and blue lines represent samples spiked
with 1.0, 0.25 and 0.05 IU mL 1, respectively, of
WHO-8 (a and c) or BAY 94-9027 (b and d).
mAbs, milli Absorbance; WHO-8, World Health
Organization 8th International Standard FVIII
concentrate.
prolonging CT (Fig. 3). These data indicate that pro-
longation of CT of BAY 94-9027 in APTT-SP assays
is likely due to interference of activation by the PEG
moiety on BAY 94-9027 on the silica matrix surface,
because neither free PEG nor PEGylated FVIII had an
effect on the CT of non-silica-based assays (e.g.
APTT-SF and PT).
FXIa-activated TGA is not affected by PEG. To fur-
ther explore the impact of the PEG moiety on silica
matrix contact activation, an FXIa-initiated TGA was
performed in FVIII-deficient plasma spiked with either
rFVIII-FS or BAY 94-9027 at 1 IU mL 1. Thrombin
generation promoted by BAY 94-9027 was similar to
rFVIII-FS, and free PEG at 30 lg mL 1 had no effect
on ETP and peak thrombin generation (Fig. 4). The
data further demonstrate that neither free PEG nor
PEGylated FVIII interferes with initiation of the clot-
ting cascade downstream of FXI, suggesting that the
PEG moiety might preclude contact activation of FXII
on the silica matrix, and that this is an in vitro
phenomenon.
Discussion
In this study, when silica-based aPTT reagents (APTT-
SP, or STA-PTT5) were used as activators in the one-
stage aPTT assay, significantly prolonged CT was
observed for PEGylated FVIII (BAY 94-9027) vs.
plasma-derived WHO-8. Comparable CTs have been
observed for BAY 94-9027 and WHO-8 when the
ellagic acid-based aPTT reagent (SynthAFax) was
used. Similar results were seen on STAGO STA analy-
ser with Cephascreen (polyphenol) or Dade Actin
(ellagic acid) as an aPTT reagent. These results dem-
onstrated that PEG modification on rFVIII molecules
does not affect FVIII:C activity in these ellagic acid-
based aPTT assays. The prolonged CT with silica
© 2014 John Wiley & Sons Ltd
 EVALUATION OF APTT ASSAYS FOR BAY 94-9027
(a)
(b)
(c)
Fig. 3. Effect of free PEG in the (a) APTT-SF, (b) APTT-SP, and (c) pro-
thrombin (PT) assays. CT, clotting time; PEG, polyethylene glycol; rFVIII-
FS, sucrose-formulated recombinant factor VIII.
activators (APTT-SP, STA PTT 5) suggests that the
PEG moiety of BAY 94-9027 may disturb silica acti-
vation of the contact pathway, presumably through
impairment of FXII activation on the silica surface.
This conclusion was supported by different clotting
assays (Figs 3 and 4). Prolonged CTs in either free
PEG- or BAY 94-9027–containing plasma were
observed only when silica-based aPTT activator was
used. There is no effect on CT or thrombin generation
in plasma spiked with free PEG or BAY 94-9027
when other activators (ellagic acid for APTT-SF, tissue
factor for PT, FXIa for TGA) were used. Our results
indicate that the prolonged CT in silica-based aPTT
assay is not specific to PEGylated FVIII.
Recently, other PEGylated therapeutic proteins have
been associated with prolonged aPTT time, including
the 200- and 400-mg dose groups of certolizumab
pegol, a PEGylated Fab fragment of a humanized anti-
tumour necrosis factor antibody [28]. However, no
association was seen between increased coagulation
© 2014 John Wiley & Sons Ltd
599
(a)
(b)
Fig. 4. Effect of free PEG on the FXIa-activated thrombin generation
assay: (a) endogenous thrombin potential (ETP) and (b) peak thrombin.
PEG, polyethylene glycol; rFVIII-FS, sucrose-formulated recombinant fac-
tor VIII.
assay time and bleeding events. Additional analyses
demonstrated that this was an in vitro phenomenon
with no effect on in vivo coagulation function. From
these studies, it was implied that the PEG moiety on
the drug interferes with the PL component of some
commercial assays [28,29] that measure aPTT,
including the HemosIL APTT-SP-liquid (Instrumenta-
tion Laboratory) and PTT-LA (Diagnostica Stago)
assays. In this report, we demonstrate that PEGylated
FVIII (BAY 94-9027) bestows normal CTs in ellagic-
based aPTT assays, PT assay and normal thrombin
generation in FXIa-initiated TGA. Therefore, the
observed prolonged aPTT clotting time with BAY 94-
9027 in the silica-based aPTT assay was also an in vi-
tro phenomenon and should not have any impact on
in vivo coagulation function.
Inhibitors to infused rFVIII are the most significant
complication for haemophilia treatment and are
detected primarily through their ability to neutralize
FVIII activity in a one-stage clotting-based assay like
the traditional Bethesda assay or the modified Nijme-
gen method. Interference by PEGylated FVIII in these
assays should be validated when using silica-based
aPTT reagents; otherwise, an ellagic acid-based aPTT
assay is highly recommended.
Haemophilia (2014), 20, 593--600
600 J-M. GU et al.

Conclusion
The one-stage aPTT method is used by most clinical
laboratories for monitoring postinfusion samples.
Among aPTT activators we used, the ellagic acid and/
or polyphenol activators are suitable for accurately
monitoring plasma levels of PEGylated FVIII in clinic.
Silica activators should be carefully scrutinized for
PEGylated therapeutics with potential for aberrant
aPTT clotting times.
Acknowledgements
Medical writing assistance was provided by Beena John, PhD, of Com-
plete Healthcare Communications, Inc. (Chadds Ford, PA, USA) and was
fully funded by Bayer HealthCare. The authors thank Andrea Loewe,
Georg Lemm, and Prasad Mathew for the helpful discussion and critically
reading the manuscript.
Author contributions
JMG, HA, JEM, VL and TM designed the study. JMG, PR, VE, LT and
LL conducted the experiments and analysed the data. JMG and TM
wrote the first draft of the manuscript. The final manuscript has been crit-
ically revised and approved by all authors.
Disclosures
All authors are employees of Bayer Health Care Pharmaceuticals and
Bayer Pharma AG.

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Haemophilia (2014), 20, 593--600 © 2014 John Wiley & Sons Ltd