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Manufacturing process used to produce long-acting recombinant factor VIII Fc fusion protein

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Recombinant factor VIII Fc fusion protein (rFVIIIFc) is a long-acting coagulation factor approved for the treatment of hemophilia A. Here, the rFVIIIFc manufacturing process and results of studies evaluating product quality and the capacity of the process to remove potential impurities and viruses are described. This manufacturing process utilized readily transferable and scalable unit operations and employed multi-step purification and viral clearance processing, including a novel affinity chromatography adsorbent and a 15 nm pore size virus removal nanofilter. A cell line derived from human embryonic kidney (HEK) 293H cells was used to produce rFVIIIFc. Validation studies evaluated identity, purity, activity, and safety. Process-related impurity clearance and viral clearance spiking studies demonstrate robust and reproducible removal of impurities and viruses, with total viral clearance >8-15 log10 for four model viruses (xenotropic murine leukemia virus, mice minute virus, reovirus type 3, and suid herpes virus 1). Terminal galactose-α-1,3-galactose and N-glycolylneuraminic acid, two non-human glycans, were undetectable in rFVIIIFc. Biochemical and in vitro biological analyses confirmed the purity, activity, and consistency of rFVIIIFc. In conclusion, this manufacturing process produces a highly pure product free of viruses, impurities, and non-human glycan structures, with scale capabilities to ensure a consistent and adequate supply of rFVIIIFc. Copyright © 2015 Biogen. Published by Elsevier Ltd.. All rights reserved.
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Manufacturing process used to produce long-acting recombinant
factor VIII Fc fusion protein
Justin McCue
*
, Rashmi Kshirsagar, Keith Selvitelli, Qi Lu, Mingxuan Zhang, Baisong Mei,
Robert Peters, Glenn F. Pierce, Jennifer Dumont, Stephen Raso, Heidi Reichert
Biogen, 14 Cambridge Center, Cambridge, MA 02142, USA
article info
Article history:
Received 6 November 2014
Received in revised form
22 April 2015
Accepted 19 May 2015
Available online xxx
Keywords:
rFVIIIFc
Human cell line
HEK 293H
Hemophilia A
Manufacturing
Recombinant
abstract
Recombinant factor VIII Fc fusion protein (rFVIIIFc) is a long-acting coagulation factor approved for the
treatment of hemophilia A. Here, the rFVIIIFc manufacturing process and results of studies evaluating
product quality and the capacity of the process to remove potential impurities and viruses are described.
This manufacturing process utilized readily transferable and scalable unit operations and employed
multi-step purication and viral clearance processing, including a novel afnity chromatography
adsorbent and a 15 nm pore size virus removal nanolter. A cell line derived from human embryonic
kidney (HEK) 293H cells was used to produce rFVIIIFc. Validation studies evaluated identity, purity,
activity, and safety. Process-related impurity clearance and viral clearance spiking studies demonstrate
robust and reproducible removal of impurities and viruses, with total viral clearance >8e15 log
10
for four
model viruses (xenotropic mu rine leukemia virus, mice minute virus, reovirus type 3, and suid herpes
virus 1). Terminal galactose-
a
-1,3-galactose and N-glycolylneuraminic acid, two non-human glycans,
were undetectable in rFVIIIFc. Biochemical and in vitro biological analyses conrmed the purity, activity,
and consistency of rFVIIIFc. In conclusion, this manufacturing process produces a highly pure product
free of viruses, impurities, and non-human glycan structures, with scale capabilities to ensure a
consistent and adequate supply of rFVIIIFc.
© 2015 Biogen. Published by Elsevier Ltd on behalf of The International Alliance for Biological
Standardization. This is an open access article under the CC BY license (
http://creativecommons.org/
licenses/by/4.0/
).
1. Introduction
Hemophilia A is an X-linked bleeding disorder, characterized by
functional factor VIII (FVIII) deciency. The mainstay treatment is
FVIII replacement therapy. Following the widespread transmission
of blood-borne viruses in the 1970s and 1980s related to the use of
plasma-derived clotting factor concentrates
[1], the FVIII gene was
cloned and recombinant protein expression and purication tech-
niques were developed. While the use of recombinant FVIII (rFVIII)
and improved purication methodology contributed to signicant
improvements in the availability and safety of FVIII replacement
therapy
[2], periodic supply shortages and manufacturing quality
breaches have continued into the 2000s. These issues reected the
relative difculty in manufacturing rFVIII, a large, multi-domain
glycoprotein with signicant post-translational modications.
To ensure the safety of rFVIII products, manufacturing processes
should be evaluated for product quality and the capacity to remove
viruses (regulations require demonstration of viral clearance using
3 viruses with differing characteristics and validation of
2 process steps that use different mechanisms for virus inacti-
vation and or/removal)
[3,4]. Additionally, processes should aim to
mitigate potential immunogenicity associated with manufacturing
rFVIII products in clonal cell lines
[5]. Manufacturing processes for
currently available rFVIII products have previously been described
[6e8]. Recombinant FVIII Fc fusion protein (rFVIIIFc; Biogen,
Abbreviations: aPTT, activated partial thromboplastin time;
a
-Gal, galactose-
a
-1,3-
galactose; BDD, B domain-deleted; FcRn, neonatal Fc receptor; FVIII, factor VIII; HC,
heavy chain; HCP, host cell proteins; HEK, human embryonic kidney; HIC, hydrophobic
interaction chromatography; ICH, International Conference on Harmonisation; IgG
1
,
immunoglobulin G
1
; MCB, master cell bank; MMV, mouse minute virus; NGNA, N-
glycolylneuraminic acid; RCB, research cell bank; Reo-3, mammalian orthoreovirus 3;
rFVIII, recombinant factor VIII; rFVIIIFc, recombinant factor VIII Fc fusion protein; RT-
PCR, reverse transcription polymerase chain reaction; SEC, size exclusion chromatog-
raphy; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SuHV-1,
suid herpes virus 1; UPLC, ultra-performance liquid chromatography; WCB, working
cell bank; X-MLV, xenotropic murine leukemia virus.
* Corresponding author. Tel.: þ1 857 756 0541; fax: þ1 617 679 2000.
E-mail addresses:
justin.mccue@biogen.com (J. McCue), rashmi.kshirsagar@
biogen.com
(R. Kshirsagar), keith.selvitelli@biogen.com (K. Selvitelli), qi.lu@
biogen.com
(Q. Lu), mingxuan.zhang@biogen.com (M. Zhang), baisong.mei@
biogen.com
(B. Mei), robert.peters@biogen.com (R. Peters), gfp555@gmail.com
(G.F. Pierce), jennifer.dumont@biogen.com (J. Dumont), stephen.raso@biogen.com
(S. Raso), heidi.reichert-robes@biogen.com (H. Reichert).
Contents lists available at ScienceDirect
Biologicals
journal homepage: www.elsevier.com/locate/biologicals
http://dx.doi.org/10.1016/j.biologicals.2015.05.012
1045-1056/© 2015 Biogen. Published by Elsevier Ltd on behalf of The International Alliance for Biological Standardization. This is an open access article under the CC BY
license (
http://creativecommons.org/licenses/by/4.0/).
Biologicals xxx (2015) 1e7
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Cambridge, MA) is the rst-approved, long-acting FVIII for adults
and children with hemophilia A for the control and prevention of
bleeding episodes, perioperative management, and routine pro-
phylaxis to prevent or reduce the frequency of bleeding episodes;
it has been designed to reduce the required infusion frequency of
prophylactic treatment
[9e12]. The presence of the Fc domain of
human immunoglobulin G
1
(IgG
1
) enables the fusion protein to bind
to the neonatal Fc receptor (FcRn), part of an endogenous intracel-
lular pathway that delays lysosomal degradation of Fc-containing
proteins (ie, IgG) by cycling them back into circulation
[13,14].Fc
fusion does not signicantly alter the higher-order structure of FVIII
or its functionality
[9,10,15]. The phase 3 A-LONG study demon-
strated an extended half-life of rFVIIIFc relative to rFVIII (~1.5-fold
increase, 19.0 h), as well as the safety and efcacy of rFVIIIFc for
the control and prevention of bleeding episodes
[11,12].
The objective of this work was to describe the rFVIIIFc
manufacturing process, and evaluate product quality and the
capacity of this process to produce a product free from viruses and
impurities.
2. Materials and methods
2.1. Manufacturing process: development of the rFVIIIFc cell line,
cell bank, and cell line characterization
The coding sequences for human FVIII and the Fc region of the
human IgG
1
(hinge and CH2 and CH3 domains) were obtained by
reverse transcription polymerase chain reaction (RT-PCR) from
human liver polyA mRNA and a human leukocyte cDNA library,
respectively. HEK 293H cells (Invitrogen, Carlsbad, CA) were stably
transfected with an expression vector containing two expression
cassettes. One cassette expressed the native human FVIII signal
sequence followed by a B domain-deleted (BDD) FVIII (S743 to
Q1638 fusion) directly linked to the Fc region of human IgG
1
with
no intervening linker. The second expression cassette held Fc with a
heterologous mouse Ig
k
B signal sequence [9,10].
Transfected HEK 293H cells were grown in serum-free medium.
Clonal cell lines were derived and the optimal cell line was selected
based on considerations for rFVIIIFc monomer productivity, rFVIIIFc
activity (measured by chromogenic assay), superior cell growth
properties, and stability. Cell lines with optimal characteristics
were then sub-cloned by limiting dilution and further character-
ized to select the production clonal cell line for manufacturing.
The clonal cell line that was selected for manufacturing was
expanded to create a research cell bank (RCB). The RCB was
expanded to create the master cell bank (MCB) from which a
working cell bank (WCB) was derived. The MCB and WCBs were
tested for identity, purity, and freedom from adventitious agents.
The transgene coding sequence, copy number, and gene integration
patterns of the MCB and a cell bank produced from a cell culture
that was propagated beyond the end of the manufacturing process
were compared based on the International Conference on Harmo-
nisation (ICH) guidelines Q5A, B and D
[16e18]. The comparison
was used to assess and conrm transgene integration and stability
of the cell line over the course of the manufacturing process.
To characterize the resultant product from this cell line, rFVIIIFc
was analyzed for the presence of two non-human glycans, terminal
galactose-
a
-1,3-galactose (
a
-Gal) and N-glycolylneuraminic acid
(NGNA).
a
-Gal was released with
a
-(1-3,4,6) galactosidase, labeled
with 2-aminobenzoic acid, and analyzed with ultra-performance
liquid chromatography (UPLC) with uorescent detection. NGNA
was released with 50 mM H
2
SO
4,
labeled with 1,2 diamino-
4,5-methylenedioxybenzene, and analyzed with UPLC with
uorescent detection. Three currently available rFVIII products
(Xyntha
®
[Wyeth Pharmaceuticals Inc, Philadelphia, PA], Advate
®
[Baxter, Westlake Village, CA], and Kogenate
®
[Bayer, Tarrytown,
NY]) were also analyzed for the presence of
a
-Gal and NGNA using
the same analytical methods.
2.2. Production of rFVIIIFc
One WCB vial was used to produce a single batch of rFVIIIFc in a
multi-step manufacturing process (Fig. 1). The inoculum preparation
phase includes thawing a WCB vial (Step 0) and expansion of culture
in shake asks (Step 1). Shake asks were then pooled and used to
inoculate the rst seed train bioreactor (for further culture expansion;
Step 2). The seed train bioreactors were operated in batch mode, with
agitation, pressure, temperature, pH, and dissolved oxygen
controlled. The expanded culture was used to inoculate a large-scale
production bioreactor (Step 3). The production bioreactor (2000 L)
was operated in fed-batch mode, during which agitation, pressure,
temperature, pH, and dissolved oxygen were controlled.
Cells and cellular debris were removed by centrifugation (Step 4)
and subsequent depth ltration steps (Step 5) to produce a claried
cell culture harvest containing the rFVIIIFc product. Detergent (Triton
X-100; Step 6) was added to the claried cell culture harvest as a
virus inactivation step. The product was then captured and puried
from the claried cell culture harvest with a FVIII-specicafnity
chromatography step using an VIIISelect column (GE Healthcare Life
Sciences, Piscataway, NJ; Step 7). VIIISelect is an immobilized re-
combinant peptide-based afnity ligand specic for FVIII that is both
highly effective and free of animal components, such as mouse
monoclonal antibodies [19]. The VIIISelect adsorbent binds to the
FVIII light chain portion of rFVIIIFc, and the product is desorbed and
collected using a buffer solution at neutral pH, to ensure the integrity
of rFVIIIFc is maintained. rFVIIIFc was further puried by charge-
based separation using anion exchange chromatography (Step 8)
followed by a ow-through anion-exchange membrane absorber
(Step 9). The rFVIIIFc product was then ltered through a 15 nm virus
lter (Planova 15N; Asahi Kasei Bioprocesses, Inc., Glenview, IL;
Step 10) to purify based on size. The virus-ltered product was
further puried using a nal chromatography step (Hydrophobic
Interaction Chromatography [HIC]; Step 11). rFVIIIFc was concen-
trated and buffer-exchanged using an ultraltration step (Step 12) to
form the bulk product. The bulk product was formulated and ltered
into bottles (Step 13) and stored at 70
C to ensure stability prior to
lyophilization and before nal lling into individual drug product
vials for use in clinical study.
2.3. Manufacturing process and impurity clearance validation
studies
Process validation studies were performed to conrm identity,
purity, quality, and activity of the rFVIIIFc product. A summary of
the analytical tests used in these assessments is shown in
Table 1.
Non-reducing sodium dodecyl sulfate polyacrylamide gel electro-
phoresis (SDS-PAGE) gels stained with colloidal Coomassie blue,
thrombin digest map, FVIII chromogenic assay and size exclusion
chromatography were also employed in validation studies for pu-
rity assessment and identity conrmation. The presence of aggre-
gated species (proteins that have undergone conformational
changes during the manufacturing or storage processes resulting in
misfolded protein species), was determined using size exclusion
chromatography (SEC)
[20]. Fc-binding activity was determined
using an FcRn binding assay
[21]. The specic activity of rFVIIIFc
was assessed using both the two-stage chromogenic substrate and
one-stage activated partial thromboplastin time (aPTT) clotting
assays [9]. Safety determination was based on testing for the
presence of bioburden and endotoxin.
J. McCue et al. / Biologicals xxx (2015) 1e72
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The purication process was designed to provide a high level of
viral clearan ce for potential adventitious viruses. To demonstrate
the capacity and robustness of the manufacturing process (and
individual steps) to remove adventitious viruses, the purication
process was evaluated for the capacity for removal of enveloped
and non-enveloped viruses using four model viruses (xenotropic
murine leukemia virus [X-MLV], mouse minute virus [MMV] ,
mammalian orthoreovirus 3 [Reo-3; also known a s reovirus
serotype 3], and suid herpes virus 1 [SuHV-1; also known as
pseudorabies virus];
Table 2). These viruses were selected to
represent ranges of physiochemical properties of several human
virus families, suc h as retroviruses, herpes viruses, reoviruses,
and parvovirus. These stu dies were conducted according to the ICH
Q5A Guidelines and the US Food and Drug Administration Points
to Con sider
[16,2 2] a nd in acc ordance with Good Laboratory
Practice
[23].
The rFVIIIFc manufacturing process was also evaluated for the
capacity to remove process-related impurities. Process-related
impurity clearance validation studies were performed both at the
manufacturing scale and in scaled-down spiking studies at the
laboratory scale. Those performed at the manufacturing scale
consisted of direct measurement of the impurities obtained from
the manufacturing process intermediates, in which clearance was
calculated from the amount removed during the process step.
Scaled-down impurity clearance validation studies involved adding
an impurity to the process intermediate and purifying the spiked
intermediate using a scaled-down chromatography step. These
studies were used when the impurity was below detectable levels
in the manufacturing process intermediates and to demonstrate
the capacity and robustness of the process to provide additional
clearance.
Table 1
Analytical tests used to assess identity, purity, activity, and safety of rFVIIIFc.
Test Method description
Polyacrylamide gel
electrophoresis
(non-reducing)
Polyacrylamide gel electrophoresis performed in the
presence of sodium dodecyl sulfate (SDS-PAGE) under
non-reducing conditions. Gels are stained using
Colloidal Coomassie Blue staining
Size exclusion
chromatography
Resolution of aggregated forms from the monomeric
form of rFVIIIFc using high performance liquid
chromatography (Sepax SRT SEC-300 column)
Coagulation activity One-stage activated partial thromboplastin time (aPTT)
clotting assay method performed in accordance with
Pharmacopeia guidelines (USP<32> and Ph. Eur. 2.7.11)
Chromogenic activity Colorimetric method performed in accordance with
Pharmacopeia guidelines (Ph. Eur. 2.7.4)
FcRn binding Neonatal Fc receptor (FcRn) competitive binding
measured using an amplied luminescent proximity
homogenous assay
Bioburden Microbial enumeration test performed in accordance
with Pharmacopeia guidelines (USP<61> and Ph. Eur.
2.6.12)
Endotoxin Kinetic turbidimetric method in accordance with
Pharmacopeia guidelines (USP<85> and Ph. Eur. 2.6.14)
FcRn, neonatal Fc receptor; Ph. Eur., European Pharmacopeia; SDS-PAGE, sodium
dodecyl sulfate polyacrylamide gel electrophoresis.
Fig. 1. Overview of the rFVIIIFc manufacturing process.
a
Viral clearance steps.
J. McCue et al. / Biologicals xxx (2015) 1e7 3
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3. Results
3.1. Cell line safety and characterization
The MCB and WCB were manufactured in accordance with
current Good Manufacturing Practice procedures, with purity,
safety, and identity test results demonstrating no detectable virus
or adventitious agents. Testing with random amplied poly-
morphic DNA, isoenzyme analysis, and RT-PCR conrmed the cell
bank origin; microbial and viral testing conrmed they were free of
bacteria, fungi, mycoplasma, and adventitious viruses. Additionally,
the cell line chosen resulted in a product free of the non-human
glycans, terminal
a
-Gal and NGNA (Table 3).
3.2. Validation studies: assessment of product quality and process
consistency
The rFVIIIFc manufacturing process generated product of
consistent purity, quality, and activity. All manufacturing steps
were successfully validated for consistency based on process per-
formance and product quality data from four batches. The
manufacturing process validation study demonstrated consistency
through evaluation of controlled parameters, in-process controls,
and product quality. Results from four validation batches are shown
in
Table 4. All batches demonstrated >97% purity, when measured
by non-reducing SEC and SDS-PAGE (
Fig. 2).
Table 4 also shows that the specic activity of rFVIIIFc was
consistent among the validation batches, with batches possessing
specic activity values of 1660e1770 IU/nmol of rFVIIIFc for the
aPTT assay and values of 1420e1720 IU/nmol for the chromogenic
substrate assay. Importantly, comparable ranges for specic activity
were achieved using both the aPTT and chromogenic substrate
assays. Additionally, results were comparable to the specic activity
of native FVIII (1429 IU/nmol)
[10] and specic activities previously
reported for rFVIIIFc (1861 ± 154 IU/nmol and 2057 ± 298 IU/nmol
using the one-stage aPTT and chromogenic substrate assays,
respectively) and that reported for ReFacto
®
(1862 IU/nmol) [9].In
addition, potency of rFVIIIFc in binding to FcRn was consistent
across the batches. No bioburden was detected in any of the
batches, and endotoxin levels were all below detectable levels.
Overall, results from analytical testing demonstrated that the
manufacturing process consistently produced a highly pure and
active rFVIIIFc product.
3.3. Validation studies: virus- and process-related impurity
clearance
Virus removal studies demonstrated signicant clearance of
viruses possessing different physical and chemical properties. The
overall total clearance for the rFVIIIFc purication process was
15.1 log
10
for X-MLV, 11.5 log
10
for SuHV-1, 8.3 log
10
for MRV-3,
and 11.9 log
10
for MMV. The detergent virus inactivation step,
the VIIISelect afnity chromatography step, the anion exchange
chromatography step, and the virus ltration step (Planova 15N)
each contributed to this viral clearance, with the most substantial
removal of model virus achieved with the use of the 15N Planova
nanolter (
Table 2).
In addition to providing robust removal of viruses, the rFVIIIFc
purication process also provided robust and reproducible
clearance of process-related impurities. Reductions in HEK 293H
host cell proteins (HCP), HEK 293H host cell DNA, and Triton X-100
are shown in
Table 5. Levels of VIIISelect ligand leachate were
below detectable levels during the manufacturing process and in
the nal product, demonstrating that only minimal, sub-detectable
levels may leach during the manufacturing process. A reduction
factor of 0.8 log
10
, obtained by performing scale-down spiking
studies of the VIIISelect ligand, further illustrates the robustness of
the rFVIIIFc manufacturing process to remove any residual
VIIISelect ligand that may be present following the VIIISelect
chromatography step. Additionally, levels of host cell DNA were
below detectable levels (<1 pg DNA/mg rFVIIIFc) in the nal
product, well below the level that is considered acceptable by the
World Health Organization
[24].
Table 2
Summary of rFVIIIFc viral clearance validation studies and virus clearance reduction factors.
Virus name Virus type Virus size
(nm)
Virus reduction factor (LRV)
Detergent
inactivation
(log
10
)
Afnity
chromatography
(log
10
)
Anion exchange
chromatography
(log
10
)
Viral ltration
(Planova 15N)
(log
10
)
Total clearance
(log
10
)
a
Xenotropic murine leukemia
virus (X-MLV)
Retrovirus 80e130 4.4
b
2.4 2.7 5.6
b
15.1
Suid herpes virus 1 (SuHV-1) Enveloped DNA
virus
120e200 4.4
b
3.1 NP 4.0
b
11.5
Reovirus type 3 (Reo-3) Non-enveloped
RNA virus
60e80 NP 2.8 NP 5.5
b
8.3
Mouse minute virus (MMV) Small DNA virus 18e22 NP >4.6
b
1.6 5.7
b
11.9
NP, not performed; log
10
, log reduction value of viral clearance.
The LRV of four viruses for the rFVIIIFc purication process steps are included.
a
Total clearance (LRV) represents the summation of the steps evaluated for viral clearance for the four viruses evaluated in the studies.
b
The > indicates virus levels were below levels of detection for the respective step.
Table 3
Levels of A) galactose-
a
-1,3-galactose (
a
-Gal) and B) N-glycolylneuraminic acid
(NGNA) in rFVIIIFc and three commercially available rFVIII products.
Sample
a
-Gal NGNA
Average %
mol/mol
(n ¼ 3)
Standard
deviation
(n ¼ 3; %)
Average
% mol/mol
(inter-day;
n ¼ 9)
a
RSD
b
(inter-day;
n ¼ 9; %)
a
rFVIIIFc <LOD
c
NA <LOD
d
NA
Xyntha 10.2 1.6 20.31 (0.73) 3.6
Advate 3.3 0.6 1.33 (0.14) 10.8
Kogenate 1.3
e
0.8 5.99 (0.32) 5.3
Positive control 41.7 0.4 ee
a
-Gal, galactose-
a
-1,3-galactose; LOD, limit of detection; NA, not applicable; NGNA,
N-glycolylneuraminic acid; rFVIIIFc, recombinant factor VIII Fc fusion protein; rFVIII,
recombinant factor VIII; RSD, relative standard deviation.
a
Analysis was done in triplicate on 3 separate days (total of n ¼ 9).
b
Inter-day (n ¼ 3 days) RSD.
c
LOD is 1.1% (0.1 pmol) for rFVIIIFc.
d
LOD is 0.28% (2.5 fmol) for rFVIIIFc.
e
1.3% is at the LOD (1.3% [0.1 pmol]) but below the limit of quantication
(2.6% [0.2 pmol]).
J. McCue et al. / Biologicals xxx (2015) 1e74
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4. Discussion
Many technologic advances have offered benets to the
hemophilia A population since FVIII was found to be enriched in the
cryoprecipitate of fresh frozen plasma in 1964
[25]. This discovery
led to the development of plasma-derived factor replacement
therapies. However, there was widespread contamination of these
products with hepatitis and HIV in the 1970s and 1980s
[1]. Viral
safety concerns accelerated the development of recombinant
clotting factors (initial approval in 1992). However, plasma proteins
added to stabilize the nal formulation of rst generation products
(eg, albumin) and plasma-derived proteins used in the cell culture
medium of second generation products continued to fuel concerns
about viral transmission (additionally, supply shortages occurred
through the early 2000s). The advent of third generation products,
free of human and animal proteins, has ushered in a new era of
theoretical safety; however, despite high theoretical safety, the
hemophilia community has remained concerned about viral
transmission. This illustrates the key importance of conducting
manufacturing validation studies in recombinant products that
demonstrate viral clearance and removal of other impurities.
The rFVIIIFc manufacturing process validation studies described
herein demonstrated the capacity of the manufacturing process to
produce a product of consistent high quality and purity and to
remove potential viruses and process-related impurities. This
process uses a number of recently developed techniques to ensure
product quality and purity, including a virus inactivation step, three
different chromatography steps, and a 15 nm pore size virus lter to
provide robust removal of viruses.
Fig. 2. A) Structural components of rFVIIIFc: the single-chain (SC) non-processed isoform and the processed isoform (FVIII light chain [A3, C1, C2] covalently linked to Fc dimer
[LCeFc/Fc]); B) Non-reducing SDS-PAGE analysis of rFVIIIFc validation batch (RECD19189-11-011) used for the determination of purity and identity. The three bands are indicative of
the three components of rFVIIIFc: FVIII heavy chain (HC), the processed isoform, and the SC isoform. Non-reducing SDS-PAGE was conducted on a 4%e12% polyacrylamide gel in
BiseTris buffer. Samples were denatured with SDS in the presence of 15 mM N-ethylmaleimide for 5 min at 75
C. The gel was stained with Colloidal Coomassie. SDS-PAGE, sodium
dodecyl sulfate polyacrylamide gel electrophoresis; MW, molecular weight; HC, FVIII heavy chain; LCeFc/Fc, FVIII light chain covalently linked to Fc dimer; SC, single-chain.
Table 5
Summary of impurity clearance achieved throughout the rFVIIIFc manufacturing
process for select process-related impurities.
Process-related impurity Impurity clearance
validation scale
Overall reduction
factor (log
10
)
a,b
HEK 293H HCP Manufacturing 5.6
HEK 293H DNA Manufacturing >7.0
VIIISelect Ligand
Leachate
Laboratory 0.8
Triton X-100 Laboratory >8.9
HCP, host cell protein; HEK, human embryonic kidney; rFVIIIFc, recombinant factor
VIII Fc.
a
Overall reduction factor (log
10
) is the sum of the reduction factor values for each
of the process steps validated for removal of the respective process-related
impurities.
b
Reduction factor ¼ log
10
(impurity load of input/impurity load of output).
Table 4
Product quality results from four validation batches.
Product attribute Test method Results
11-011 11-012 11-013 11-014
Identity Non-reducing SDS-PAGE; Comparable to reference standard
Thrombin digest map Conforms to reference standard
FVIII chromogenic substrate assay Meets biologic activity specication
Purity Non-reducing gel electrophoresis (%) 97.7 98.1 98.2 98.6
Size exclusion chromatography (%) >99.0 >99.0 >99.0 >99.0
Activity Coagulation activity based on one-stage
aPTT clotting assay specic activity
(IU/nmol rFVIIIFc)
a
1660 1700 1770 1660
Activity based on chromogenic substrate
assay specic activity (IU/nmol rFVIIIFc)
a
1420 1620 1640 1720
FcRn binding relative potency
b
(%) 127 127 120 122
Safety Bioburden (CFU/10 mL) 0 0 0 0
Endotoxin (EU/mL) <1.00 <1.00 <1.00 <1.00
aPTT, activated partial thromboplastin time; CFU, colony-forming units; ELISA, enzyme-linked immunosorbent assay; EU, endotoxin units; FcRn, neonatal Fc receptor; rFVIIIFc,
recombinant factor VIII Fc; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; WHO, World Health Organization.
a
Coagulation activity is calibrated against the WHO international standard for FVIII. For comparison, the specic activity of rFVIII is 1429e1862 [9,10].
b
One GMP batch manufactured using the same process, scale, and facility has been designated as a reference standard. Potency was determined against this reference
standard.
J. McCue et al. / Biologicals xxx (2015) 1e7 5
Please cite this article in press as: McCue J, et al., Manufacturing process used to produce long-acting recombinant factor VIII Fc fusion protein,
Biologicals (2015), http://dx.doi.org/10.1016/j.biologicals.2015.05.012
A key feature of rFVIIIFc's manufacturing process is the use of a
human cell line and a process that is free from added human-
or animal-derived components. The HEK 293H cell line has
biochemical properties that are advantageous for recombinant
protein expression, such as amenability to transfection, high
efciency, and effective translation of human protein processing
and production
[26e28]. This cell line has been selected to produce
a number of human recombinant protein therapeutics, including
drotrecogin alfa-activated protein C, a factor VIII product, and a
factor IX product
[5,27e31]. Another advantage of using a human
cell line is this ensures no non-human glycan structures are
incorporated into the expressed proteins, which need to be moni-
tored and screened for during cell line development in cells lines
derived from rodents [32,33]. Results of this work indicate that the
use of a human cell line to manufacture rFVIIIFc yields a product
free of the non-human glycan structures found in proteins
expressed in hamster cell lines. It has been previously reported that
all humans possess anti-NGNA antibodies and sometimes at high
levels, approaching 0.1%e0.2% of circulating IgG
[34]. Anti-
a
-Gal
antibodies (IgE) have also been previously observed in humans
[35]. As a result,
a
-Gal and NGNA are potentially immunogenic. In
this analysis, neither NGNA nor
a
-Gal were detected in rFVIIIFc, but
different amounts of both were found in the three commercially
available rFVIII products: Advate, Xyntha, and Kogenate (
Table 3),
all produced with hamster cell lines. Although the impact of these
antigens in vivo is not known, their absence may result in lower
immunogenicity
[5].
Similar viral clearance steps that include multiple chromatog-
raphy steps, a virus ltration step, and a virus inactivation step have
also been utilized to manufacture other rFVIII products. To our
knowledge, this is the rst reported use of a 15 nm nanolter in the
manufacturing process of a rFVIII product. The viral clearance
resulting from the manufacturing process of rFVIIIFc can be
compared with that of another rFVIII product produced in
mammalian (CHO) cells
[6]. The CHO-based rFVIII process achieved
>11.4 logs for X-MLV, >14.0 logs for SuHV-1, >10.3 logs for Reo-3,
and 5.2 logs for MMV. The process removed viruses to below
detectable levels for three of the four model viruses evaluated in
the studies (X-MLV, SuHV-1, and Reo-3). However, the process did
not clear MMV to below detectable levels, achieving an overall
clearance of 5.2 logs compared with >11.9 logs achieved using the
rFVIIIFc manufacturing process. This may be due to the use of a
larger (35 nm) pore size virus lter in the rFVIII manufacturing
process, which was less effective at removing relatively small MMV
viruses. MMV is a surrogate for parvoviruses, among the smallest
human pathogens.
Reducing the pore size in nanoltration is known to greatly
enhance the effectiveness of viral clearance without affecting
puried FVIII
[36]; the small pore size (15 nm) of the Planova 15N
virus lter provides an effective barrier for a wide range of large-
size impurities. In the current study, the Planova 15N lter pro-
vided an extremely stringent level of clearance providing a total
reduction factor 8.3 log
10
for each of the model viruses tested and
>15 log
10
clearance of retroviruses. Overall, these results demon-
strate that the rFVIIIFc manufacturing process can effectively clear
retroviruses, in addition to a broad spectrum of adventitious virus
types.
5. Conclusions
Over more than two decades, the safety of manufactured rFVIII
proteins has improved dramatically compared with the previous
manufacture of plasma-derived FVIII, but has not been without
challenges. The rFVIIIFc manufacturing process employs multiple
new methods including a unique cell line and state-of-the-art
purication and viral ltration to consistently produce a novel, fully
active, and highly puried product free from viral contaminants or
impurities. Importantly, by utilizing a scalable and transferable
process, the product can be produced within any of the manufac-
turer's large-scale manufacturing facilities, reducing supply risks
[37].
Author contributions
Justin McCue composed the manuscript. Stephen Raso analyzed
the data. All authors contributed to the interpretation of the data,
manuscript revisions, and approval of the submitted version.
Role of funding source
Financial support for the conduct of the research and prepara-
tion of the manuscript were provided by Biogen.
Declaration of interest
All authors were employees of and held equity interest in Biogen
at the time this research was conducted.
Acknowledgments
Editorial support for this manuscript was provided by Samantha
Taylor, PhD, of Evidence Scientic Solutions and Laurie Orloski,
PharmD, of MedErgy, and was funded by Biogen.
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Biologicals (2015), http://dx.doi.org/10.1016/j.biologicals.2015.05.012
... The discovery in 1964 that fresh frozen plasma cryoprecipitate contained high levels of FVIII gave rise to plasma-derived factor replacement therapy [7]. These concentrates have significantly improved the quality of life of hemophilic patients by reducing the number of bleeds and their consequences, such as hemophilic arthropathy. ...
... Following these events, coagulation factor concentrates of recombinant origin were developed in the 1990's. The first of a long series was marketed in 1992 [7], while the second and third generations of recombinant products were then added to the first generation of rFVIII, with a superior efficacy and safety profile [7]. ...
... Following these events, coagulation factor concentrates of recombinant origin were developed in the 1990's. The first of a long series was marketed in 1992 [7], while the second and third generations of recombinant products were then added to the first generation of rFVIII, with a superior efficacy and safety profile [7]. ...
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Background Prophylactic factor replacement in patients with hemophilia B improves outcomes but requires frequent injections. A recombinant factor IX Fc fusion protein (rFIXFc) with a prolonged half-life was developed to reduce the frequency of injections required. Methods We conducted a phase 3, nonrandomized, open-label study of the safety, efficacy, and pharmacokinetics of rFIXFc for prophylaxis, treatment of bleeding, and perioperative hemostasis in 123 previously treated male patients. All participants were 12 years of age or older and had severe hemophilia B (endogenous factor IX level of ≤2 IU per deciliter,or ≤2% of normal levels). The study included four treatment groups: group 1 received weekly dose-adjusted prophylaxis (50 IU of rFIXFc per kilogram of body weight to start), group 2 received interval-adjusted prophylaxis (100 IU per kilogram every 10 days to start), group 3 received treatment as needed for bleeding episodes (20 to 100 IU per kilogram), and group 4 received treatment in the perioperative period. A subgroup of group 1 underwent comparative sequential pharmacokinetic assessments of recombinant factor IX and rFIXFc. The primary efficacy end point was the annualized bleeding rate, and safety end points included the development of inhibitors and adverse events. Results As compared with recombinant factor IX, rFIXFc exhibited a prolonged terminal halflife (82.1 hours) (P<0.001). The median annualized bleeding rates in groups 1, 2, and 3 were 3.0, 1.4, and 17.7, respectively. In group 2, 53.8% of participants had dosing intervals of 14 days or more during the last 3 months of the study. In groups 1, 2 and 3, 90.4% of bleeding episodes resolved after one injection. Hemostasis was rated as excellent or good during all major surgeries. No inhibitors were detected in any participants receiving rFIXFc; in groups 1, 2, and 3, 73.9% of participants had at least one adverse event, and serious adverse events occurred in 10.9% of participants. These events were mostly consistent with those expected in the general population of patients with hemophilia. Conclusions Prophylactic rFIXFc, administered every 1 to 2 weeks, resulted in low annualized bleeding rates in patients with hemophilia B
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Mammalian cell expression systems are the dominant tool today for producing complex biotherapeutic proteins. In this chapter, we discuss the basis for this dominance, and further explore why the Chinese hamster ovary (CHO) cell line has become the prevalent choice of hosts to produce most recombinant biologics. Furthermore, we explore some of the innovations that are currently in development to improve the CHO cell platform, from cell line specific technologies to overarching technologies that are designed to improve the overall workflow of bioprocess development. Graphical Abstract
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