BAY 81‐8973, a full‐length recombinant factor VIII: manufacturing processes and product characteristics

BAY 81‐8973 (Kovaltry®, Bayer, Berkeley, CA, USA) is an unmodified, full‐length recombinant human factor VIII (FVIII) approved for prophylaxis and on‐demand treatment of bleeding episodes in patients with haemophilia A. The BAY 81‐8973 manufacturing process is based on the process used for sucrose‐formulated recombinant FVIII (rFVIII‐FS), with changes and enhancements made to improve production efficiency, further augment pathogen safety, and eliminate animal‐ and human‐derived raw materials from the production processes. The baby hamster kidney cell line used for BAY 81‐8973 was developed by introducing the gene for human heat shock protein 70 into the rFVIII‐FS cell line, a change that improved cell line robustness and productivity. Pathogen safety was enhanced by including a 20‐nm filtration step, which can remove viruses, transmissible spongiform encephalopathy agents and potential protein aggregates. No human‐ or animal‐derived proteins are added to the cell culture process, purification or final formulation. The BAY 81‐8973 manufacturing process results in a product of enhanced purity with a consistently high degree of sialylation of N‐linked glycans on the molecular surface. The innovative manufacturing techniques used for BAY 81‐8973 yield an effective rFVIII product with a favourable safety profile for treatment of haemophilia A.


Introduction
Haemophilia A is a congenital blood coagulation disorder resulting from mutations in the F8 gene [1,2]. Patients with severe haemophilia A have levels of clotting factor VIII (FVIII) that are <1% of normal, placing them at risk for spontaneous bleeding episodes [1,3]. These bleeding episodes can be controlled when they occur, with on-demand infusion of a FVIII product, or they can be prevented with FVIII infusions given prophylactically [1].
Commercial production of recombinant FVIII (rFVIII) for treatment of haemophilia A is a challenging process, given the complex nature of the FVIII protein [4]. The first rFVIII products were introduced in the early 1990s; these products used human serum albumin and animal-derived materials in the manufacturing process [4]. Refinements in rFVIII manufacturing have reduced or eliminated use of human-or animal-derived materials, which decreased the potential for pathogen transmission [4].
BAY 81-8973 (Kovaltry â , Bayer, Berkeley, CA, USA) is an unmodified, full-length recombinant human FVIII that has the same FVIII amino acid sequence as the currently marketed product sucroseformulated rFVIII (rFVIII-FS; Kogenate â FS; Bayer) and is manufactured using innovative techniques [5]. The principal reasons for the development of a new rFVIII manufacturing process were to eliminate human-and animal-derived raw materials from the manufacturing processes, optimize the production process by reducing the number of manufacturing steps, further augment pathogen safety, and develop a new manufacturing platform using the latest technologies.

BAY 81-8973 manufacturing process
The BAY 81-8973 manufacturing process is a result of modifications and enhancements to the manufacturing process used for rFVIII-FS (Table 1, Fig. 1) [6], which itself is based on the manufacturing process used for Bayer's first marketed rFVIII (Kogenate â , Bayer) [7]. A comparison of the principal manufacturing steps for BAY 81-8973 and those for rFVIII-FS and other marketed rFVIII products, including prolonged half-life products, is shown in Table 2.

Development of the protein-free media for cell culture
Recombinant FVIII products are produced in mammalian cell cultures because of the large size of the FVIII protein and the high degree of posttranslational modifications [7]. Development of a cell culture medium free of human-or animal-derived raw materials is critical to minimizing the risk of pathogen contamination from these sources [7,8].
The cell culture medium used for rFVIII-FS contains human plasma protein solution (HPPS) and recombinant insulin but no proteins derived from animal sources [9]. The cell culture medium used for BAY 81-8973 was modified to eliminate HPPS, resulting in a medium free of human-and animalderived raw materials [5]. Ethylenediaminetetraacetic acid (EDTA)/FeSO 4 and a trace metals solution are added to replace the iron and trace metals supplied by HPPS [8]. To replace the shear-protectant function of HPPS, Pluronic â F-68 (BASF, Florham Park, NJ, USA) is added. Pluronic â F-68 is a polyol copolymer that acts as a surfactant, preventing cell damage by allowing cells to drain away from bubbles formed in bioreactors during stirring or sparging of cell cultures [8]. In addition, the BAY 81-8973 manufacturing process uses a 3-(N-morpholino)propanesulphonic acid (MOPS)-histidine buffer instead of sodium bicarbonate to reduce pCO 2 levels in the cell culture [10,11]. The final composition of the BAY 81-8973 protein-free cell culture medium was shown to enhance the specific rFVIII productivity of the baby hamster kidney (BHK) cells used to produce BAY 81-8973 [8].

Cell culture and isolation processes
BAY 81-8973 is manufactured using the same cell expression system (BHK-21) as rFVIII-FS but with a more robust, improved productivity cell line [5,6]. The cell line was developed through introduction of the gene for human heat shock protein 70 (HSP70) [5,12] into the cell line used for manufacture of rFVIII-FS. HSP70 is a chaperone protein that may increase FVIII expression by facilitating proper protein folding and improves cell survival by inhibiting apoptosis (ie, programmed cell death) [5,13,14].
Continuous cell separation and membrane-based ion exchange chromatography rapidly capture and concentrate rFVIII from the cell culture harvest [5,6,15]. Isolating the sensitive FVIII molecule as quickly as possible from the cell culture harvest helps ensure product quality and improves the robustness and efficiency of the manufacturing process [6]. After cells and cellular debris are removed from the harvest through a continuous cell separation process using disposable filters, a large-scale ion-exchange membrane chromatography process captures the intact FVIII molecule at least 10-fold faster than the previously used conventional chromatography processes [6]. The FVIII in the membrane chromatography eluate is then stabilized as an intermediate for long-term storage.

Purification process
Immunoaffinity, metal affinity and ion exchange chromatography are used to purify BAY 81-8973 and • Same process, presentation, excipients, and fill sizes • Same process, presentation, excipients, and fill sizes remove process-and product-related impurities. The BAY 81-8973 purification process was optimized from the rFVIII-FS process through the elimination of gelatin sepharose chromatography and replacement of a polishing chromatography step with a more efficient disposable membrane adsorber capsule for residual DNA removal [5,6]. The purification process also includes a 20-nm viral filtration step, which provides robust and orthogonal virus clearance. No human-or animal-derived raw materials are added in the purification process [5].

Viral inactivation and removal
The BAY 81-8973 manufacturing process includes a highly effective and robust enveloped virus inactivation step using a detergent solution in the unpurified intermediate FVIII product [16]. This efficient, stateof-the-art manufacturing process is integrated into the elution and bagging process of the FVIII intermediate product, ensuring a physical process segregation that further protects the product from potential viral contamination [6]. Viral clearance is enhanced using a 20-nm pore size viral filter (pore size is limited by the size of the full-length rFVIII protein) capable of removing viruses and transmissible spongiform encephalopathy (TSE) agents as well as potential protein aggregates [5,6,16]. The downstream BAY 81-8973 manufacturing process was validated for viral clearance capability using four model viruses and scaled-down systems representative of current Good Manufacturing Practice (cGMP) steps. The model viruses were xenotropic murine leukaemia virus (X-MuLV), pseudorabies virus (PRV), porcine parvovirus (PPV) and reovirus type 3 (Reo3). The viral clearance capability of the manufacturing process was determined by evaluating the ability of each process step to reduce viral infectivity, as measured using a log 10 reduction factor (LRF), in which the amount of model virus introduced before the process step was compared with the amount of virus remaining after the process step. Virus infectivity was determined using cytopathiceffect-based 50% tissue culture infective dose (TCID 50 ) assays.
Under the worst-case scenario, the total number of endogenous retrovirus-like particles in the clarified tissue-culture-fluid harvest material used to produce a 4000-IU dose is approximately 10 7.32 particles per   Haemophilia (2017), 23, e67--e78 dose (7.32 log 10 ). Thus, the significant retrovirus clearance by the BAY 81-8973 manufacturing process provides a high safety margin of ≥9.80 log 10 with a naive capture membrane adsorber and column resins and ≥6.40 log 10 for reused capture membrane adsorber and resins. The BAY 81-8973 manufacturing process has also been demonstrated to remove potential contamination from TSE agents. Results from in vivo studies using hamster-adapted Scrapie strain 263K as the TSE agent have shown that the viral filtration step significantly reduced TSE infectivity in hamsters (LRF of 3.25 and 4.29, respectively, for duplicate filtration experiments).

Final formulation
BAY 81-8973 is available in the same vial sizes (250, 500, 1000, 2000, 3000 IU) as rFVIII-FS [17]. BAY 81-8973 contains the same excipients in the same quantities and has the same temperature stability and shelf-life as rFVIII-FS. The recommended storage conditions for BAY 81-8973 are +2°C to +8°C (36°F-46°F) for up to 30 months from the date of manufacture [17]. Within this period, BAY 81-8973 can be stored for up to 12 months at temperatures up to +25°C (77°F) [17].

BAY 81-8973 characteristics
The manufacturing process used for BAY 81-8973 results in an rFVIII product of enhanced purity with a consistently high degree of sialylation of N-linked glycans on the molecular surface, a posttranslational modification step that is important to the half-life of some mammalian proteins [5,18,19]. The BAY 81-8973 molecule has been characterized in detail. Assays for monitoring critical quality attributes are routinely used for release of product lots against specifications. Extended characterization has also been performed to support the body of knowledge and licensing submission.

Protein backbone profile
The BAY 81-8973 protein is synthesized as a singlechain 330-kDa precursor with a domain structure of A1-A2-B-A3-C1-C2 subunits. Proteolytic processing yields an A1-A2-B heavy chain and A3-C1-C2 light chain to form a large heterodimeric structure linked by a divalent cation bridge (Fig. 2). The dimer of heavy and light chains has a combined average molecular mass of 264,723 Da, composed of 2332 amino acids. Glycosylation of the molecule increases the molecular mass to~330,000 Da. Further processing leads to a degree of cleavage within the nonactive B domain, as is observed for all full-length FVIII molecules. The FVIII molecule has eight disulphide bonds [4], and these have been confirmed to be as expected using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of tryptic digests. The two assays routinely used for monitoring lotto-lot production protein profile consistency are size exclusion chromatography (SEC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).

Size exclusion chromatography
The principal components of rFVIII products are separated into peaks by SEC, and the peaks are quantified based on their fluorescence emission. The SEC procedure separates the components according to their hydrodynamic volume, or molecular size. To identify the components present across the BAY 81-8973 SEC profile, fraction collection was performed across the separations. Collected fractions were subjected to potency testing, SDS-PAGE silver staining, Western blot analysis, and mass spectrometry to confirm the identity of the components within each reported SEC peak area (data not presented). A typical SEC profile for BAY 81-8973 compared with that for rFVIII-FS is shown in Fig. 3. The highmolecular-weight region 1 peak observed in the rFVIII-FS molecule predominantly contains plasma proteins derived from the HPPS added to the cell culture media for that product. This peak is minimized in the BAY 81-8973 molecule owing to the absence of plasma proteins and the presence of the viral filter, which is capable of removing particles >20 nm in size, at the final stage of the purification process. The main SEC peak (region 2) contains the heterogeneous glycoforms of the intact BAY 81-8973 molecule. Region 2 also contains active rFVIII that has been partially cleaved in the C-terminal region of the heavy chain B domain. Proteolytic processing of the inactive B domain is known to occur in all FVIII products, including plasma-derived products (primarily at Arg 1313 ) [20]. All cleaved fragments of the molecule that contain the highly glycosylated B domain elute within this main peak. The region 3 peak contains active BAY 81-8973, primarily identified as the fully B-domain-deleted material. The region 4 peak contains no active material and consists of noncomplexed (free) light chain, the productrelated impurity. In general, levels of high-molecularweight impurities and protein aggregates are below the limit of quantitation.

Electrophoretic analysis
SDS-PAGE analysis is used to identify the product relative to other rFVIII products manufactured on site and to calculate purity. Typical SDS-PAGE profiles are presented in Fig. 4. The heterogeneous heavychain profile covers the range from~200 to 90 kDa, with the fully B-domain-deleted heavy chain at 90 kDa. The light chain of the molecule is observed at 80 kDa. The C-terminus of the B domain elutes as a diffuse band between 50 and 60 kDa. This region, including any other impurity bands that could theoretically appear, is quantified and subtracted from the reported purity of the molecule. This step is performed for historical tracking purposes rather than as a result of the C-terminus of the B domain being considered an impurity. Haptoglobin bands are identified in the rFVIII-FS product.

Post-translational modifications
Multiple N-linked and O-linked glycosylation sites are present on the BAY 81-8973 structure, predominantly within the B domain of the molecule (Fig. 2). As part of routine release testing, glycan consistency and sialylation are recorded for each lot of BAY 81-8973 using an oligosaccharide map.
Released glycans are labelled with a fluorescent label and separated into peaks using high-performance liquid chromatography in normal phase/ion exchange mixed mode, with fluorescence detection. This procedure separates the labelled components primarily according to their ionic charge, which is dependent on the number of sialic acid residues covalently bound to the glycans, and also by their size and structure.
The peptide-N-glycosidase F (PNGase F)-released oligosaccharide map obtained for the N-linked glycans of BAY 81-8973 product reference standard, with the profile observed for the rFVIII-FS lot presented for comparison, is shown in Fig. 5. BAY 81-8973 shows a relative increase in the highly sialylated branched structures. This high level of terminal sialylation is quantified and measured against a Further characterization to confirm the specific glycan structures and sites was performed using LC-MS analysis of both tryptic-and thrombin-digested samples. Additionally, characterization of the terminal sialylation of the BAY 81-8973 glycans was performed, following sialidase digestion release, to confirm that N-glycolylneuraminic acid (NGNA) and a-galactoselinked sialic acid were consistently below the quantitation limit (<1%).
Tyrosine sulphation is a post-translational modification common to blood coagulation proteins. BAY 81-8973, as expected, has six highly occupied tyrosine sulphation sites at residue 346 in the A1 domain; residues 718, 719, and 723 in the A2 domain; and residues 1664 and 1680 in the light chain, as well as a very low occupancy sulphation site at tyrosine 395 (Fig. 2). These specific sites were confirmed through analysis of tryptic peptide maps and can also be seen on LC-MS profiles of BAY 81-8973 digested with thrombin.

Pharmacokinetics, efficacy and tolerability in clinical trials
In clinical trials, the pharmacokinetic profile of BAY 81-8973 was noninferior to that of rFVIII-FS, and for some variables, BAY 81-8973 showed more favourable pharmacokinetics [18]. Compared with rFVIII-FS, BAY 81-8973 had a longer half-life, higher area under the curve, longer mean residence time and slower clearance [18].
In studies of previously treated children, adolescents, and adults with severe haemophilia A, BAY 81-8973 was efficacious when administered as prophylaxis, either 2 or 3 times weekly (or up to every other day in patients ≤12 years old), and as on-demand treatment [21][22][23]. The incidence of treatment-related adverse events in each individual study was ≤7%, and no FVIII inhibitors developed [21][22][23].
The principal clinical trials for BAY 81-8973 and other marketed rFVIII products are summarized in Table 5. All products have demonstrated efficacy, safety and tolerability in patients with severe haemophilia A. In clinical trials, BAY 81-8973 prophylaxis was efficacious in previously treated patients using either a standard-dose thrice-weekly prophylaxis regimen or a lower-dose twice-weekly regimen [21,22]. Twice-weekly BAY 81-8973 prophylaxis may be appropriate for patients whose bleeding episodes currently are well-controlled on a twice-weekly regimen and for patients with severe haemophilia A who have a mild bleeding phenotype. In the LEOPOLD clinical trial program, a mild bleeding phenotype and good   Haemophilia (2017), 23, e67--e78 SPINART [40] Valentino et al. [41] Recht et al. [42] Guardian 1 [43] GENA-08 [44] AFFINITY [45] A-LONG [46] PROLONG-ATE     response to twice-weekly prophylaxis was associated with older age (≥30 years), few target joints, low number of joint bleeds, high von Willebrand factor levels (≥120%) and high FVIII trough levels at 72 hours postinfusion [16,24,25]. Other considerations in determining the appropriateness of a twiceweekly regimen include the patient's activity level, level of adherence to prophylaxis and FVIII pharmacokinetic profile. Patients currently receiving prophylaxis three times weekly may be candidates for twiceweekly dosing if they have difficulty adhering to the more frequent regimen, depending on their history of bleeding frequency.

Conclusions
BAY 81-8973 is an unmodified, full-length recombinant human FVIII approved for treatment of haemophilia A. BAY 81-8973 has the same FVIII amino acid sequence as rFVIII-FS and is manufactured using innovative manufacturing techniques. Characterization data show that the molecule has the expected profiles for protein size distribution and sequence, including the expected disulphide bonds. Aggregate levels are lower in the BAY 81-8973 product relative to those in rFVIII-FS, primarily owing to the removal of plasma proteins. The posttranslational modification sites and structures are confirmed, and high levels of sialylation of the branched N-glycans have been shown. This profile could potentially translate to benefits for the patient. The BAY 81-8973 manufacturing process results in a product of enhanced purity that has been shown to be efficacious and well-tolerated for prophylaxis and on-demand treatment of bleeding episodes in patients with severe haemophilia A.