Search

Recombinant therapeutic proteins are typically produced through cell culture process. Host cell proteins (HCPs) are endogenous proteins derived from the host cells used for such bioproduction. HCPs form a major class of process-related impurities and even at low levels they can potentially compromise the safety and efficacy of biopharmaceuticals. Therefore, they need to be adequately removed via the downstream process. HCPs are complex mixtures with diverse physiochemical properties, and certain subpopulations can bind to the intended product. Hence reducing them to the generally accepted level can be challenging. This article reviews effective HCP removing strategies at different stages of downstream process for monoclonal antibodies and Fc-fusion proteins. When used in combination, these strategies can greatly enhance the chance of meeting the drug substance specifications for residual HCP.

Introduction Recombinant factor VIII (rFVIII) products with extended half-lives have the potential to improve adherence and outcomes in haemophilia beyond the results obtained with conventional rFVIII products. Aim In the absence of head-to-head comparisons, annualized bleed rates (ABRs) and weekly factor consumption with rFVIII Fc fusion protein (rFVIIIFc) and conventional rFVIII products were indirectly compared using studies of continuous prophylaxis. Methods A systematic literature review was conducted to identify studies of rFVIII products for comparison with rFVIIIFc in the continuous prophylactic treatment of previously treated adolescents and adults with moderate and severe haemophilia A. Mean ABRs were compared between rFVIIIFc and individual rFVIII studies and between rFVIIIFc and a pooled measure for rFVIII estimated by meta-analysis. Comparisons of factor consumption were based on mean or median weekly factor consumption. Results Results from seven studies of conventional rFVIII products (injections 2–4 times week−1) were compared with rFVIIIFc (injections 1.4–2.4 times week−1). The pooled mean ABR for rFVIII products was significantly higher compared with rFVIIIFc (difference = 2.0; P = 0.007). Compared with most rFVIII studies, the reported weekly factor consumption was lower with rFVIIIFc [mean differences = 15.5–21.8 IU kg−1 week−1 (17–26%); median differences = 12.7–29.8 IU kg−1 week−1 (16–37%)]. In one comparison, mean weekly factor consumption with rFVIII was significantly lower but mean ABR was significantly higher than rFVIIIFc. Conclusion Prophylaxis with rFVIIIFc may be associated with improved bleeding rates and lower weekly factor consumption than more frequently injected rFVIII products. Relative to rFVIII products with similar bleeding rates, results indicate that rFVIIIFc is associated with reduced weekly factor consumption while requiring fewer prescribed injections.

SummaryThe safety, efficacy, and prolonged half-life of recombinant factor IX Fc fusion protein (rFIXFc) were demonstrated in the Phase 3 B-LONG (adults/adolescents ≥12 years) and Kids B-LONG (children Supplementary Material to this article is available online at www.thrombosis-online.com.

Introduction PBPK models incorporate complex drug and system (ie, physiology) information into a mathematical framework to support dose selection while considering various patient factors (eg, age, sex, and comorbidity). Through "predict-learn-confirm cycles," PBPK models enable dose prediction to optimize clinical study design. BIVV001 (rFVIIIFc-VWF-XTEN), a new class of FVIII replacement therapy designed to break the von Willebrand factor (VWF) half-life ceiling (Chhabra et al.Blood. 2020; Konkle et al.Blood. 2018), consists of a single recombinant FVIII fused to dimeric Fc (to permit recycling of BIVV001 by a naturally occurring pathway via Fc neonatal receptors [FcRn]), a VWF D′D3 domain, and two XTEN® polypeptides (XTEN® is a registered trademark of Amunix Pharmaceuticals, Inc.). In a Phase 1 study (Lissitchkov et al.Blood. 2019), four once-weekly BIVV001 50 IU/kg infusions provided high sustained FVIII activity with a mean half-life of 41.3 hours. We aimed to build and verify an initial PBPK model to characterize BIVV001 activity levels in adults with severe hemophilia A. Methods To our knowledge, this is the first time that a PBPK model has been studied in hemophilia. We assumed that the distribution of BIVV001 and recombinant FVIII Fc fusion protein (rFVIIIFc) (Eloctate®) involves the FcRn recycling pathway that is similar to monoclonal antibodies. First, to evaluate the feasibility of the modeling approach, we built a PBPK model (Simcyp, Sheffield, UK) that captured FcRn-dependent recycling of Fc fusion proteins. Model input parameters were adjusted to capture observed rFVIIIFc activity levels with a prediction error of Results To fit the BIVV001 data, the PBPK model input parameters, KD1, vascular reflection coefficient (σv), and Kup, were modified compared with those used for the rFVIIIFc PBPK model. The PBPK model captured observed BIVV001 exposure with a low prediction error ( Conclusions We have developed a PBPK model that accurately predicts the high sustained FVIII activity levels observed with BIVV001 exposure in adults with severe hemophilia A. The PBPK model framework can be a useful tool in the development of FVIII replacement therapies for hemophilia A. Disclosures Xia: Sanofi:Current Employment, Current equity holder in publicly-traded company.Katragadda:Sanofi:Current Employment, Current equity holder in publicly-traded company.Teutonico:Sanofi:Current Employment, Current equity holder in publicly-traded company.Macha:Sanofi:Current Employment, Current equity holder in publicly-traded company.Demissie:Sanofi:Current Employment, Current equity holder in publicly-traded company.Benson:Sanofi:Current Employment, Current equity holder in publicly-traded company.

Introduction Monitoring of factor IX (FIX) replacement therapy in haemophilia B relies on accurate coagulation assays. However, considerable interlaboratory variability has been reported for one‐stage clotting (OSC) assays. This study aimed to evaluate the real‐world, interlaboratory variability of routine FIX activity assays used in clinical haemostasis laboratories for the measurement of recombinant FIX Fc fusion protein (rFIXFc) activity. Methods Human FIX‐depleted plasma was spiked with rFIXFc at 0.80, 0.20 or 0.05 IU/mL based on label potency. Participating laboratories tested samples using their own routine OSC or chromogenic substrate (CS) assay protocols, reagents and FIX plasma standards. Laboratories could perform more than one measurement and method, and were not fully blinded to nominal activity values. Results A total of 142 laboratories contributed OSC results from 175 sample kits using 11 different activated partial thromboplastin time (aPTT) reagents. The median recovered FIX activity for the 0.80, 0.20 and 0.05 IU/mL samples was 0.72 IU/mL, 0.21 IU/mL and 0.060 IU/mL, respectively. Across all OSC reagents, interlaboratory variability (% CV) per aPTT reagent ranged from 9.4% to 32.1%, 8.2% to 32.6% and 12.2% to 42.0% at the 0.80, 0.20 and 0.05 IU/mL levels, respectively. CS results showed excellent median recoveries at all nominal levels (87.5% to 115.0%; n = 11) with low interlaboratory variability (CV 3.6% to 15.4%). Conclusion This large, real‐world data set indicates that rFIXFc activity in plasma samples can be accurately measured with the majority of routine OSC and CS assay methods. Given the variation in FIX assay procedures between sites, it is important that individual laboratories qualify their in‐house methods for monitoring of rFIXFc activity.

Fc fusion proteins and Fc fusion peptides or peptibodies are chimeric molecules composed of an active pharmacological protein or peptide and the Fc fragment of an immunoglobulin. The primary aim of this drug construct is to prolong the half-life of the active component. This molecular architecture is seen in drugs, such as etanercept, romiplostim, and the recombinant factor VIII (efmoroctocog alfa). A considerable number of Fc fusion proteins and peptibodies are currently in pre-clinical and clinical development. The isolated effect of the Fc fragment has been studied intensively during last years, but is still poorly understood in the clinical setting and in relation with the active drug and underlying disease. In this short review, we will propose new hypotheses of possible immunomodulatory functions of the Fc fragment of romiplostim in patients with primary immune thrombocytopenia.

High-mannose-type glycans (HMGs) are aberrantly enriched on HIV envelope glycoproteins. However, there is currently no drug selectively targeting HIV-associated HMGs. Here, we describe a novel HMG-targeting “lectibody,” a recombinant Fc-fusion protein comprising human IgG1 Fc and a novel actinohivin lectin variant (Avaren) obtained by structure-guided modifications for improved overall surface charge properties (AvFc). AvFc was engineered and produced using a rapid and scalable plant-based transient overexpression system. The lectibody exhibited potent antiviral activity against HIV-1 groups M and O primary viruses, as well as HIV-2 and simian immunodeficiency virus (SIV) strains, without affecting normal human blood cells. Furthermore, the lectibody induced Fc-mediated cell killing activity against HIV-1-infected cells and selectively recognized SIVmac239-infected macaque mesenteric lymph node cells in vitro. AvFc showed an extended serum half-life in rats and rhesus macaques, while no discernible toxicity was observed upon repeated systemic dosing in mice. These results highlight AvFc’s potential as a biotherapeutic targeting HIV-associated HMGs of cell-free virions, as well as productively infected cells, providing a foundation for new anti-HIV strategies. Efficient and cost-effective bioproduction in greenhouse facilities may open unique possibilities for further development of AvFc., High-mannose-type glycans are clustered on HIV envelope, but there is currently no drug targeting these glycans. Matoba and colleagues developed a novel lectin-Fc fusion protein, or “lectibody,” selectively recognizing this glycobiomarker, which exhibited potent HIV neutralization and Fc-mediated HIV+ cell-killing activities without affecting uninfected normal cells.

The long-acting growth hormone (LAGH) is a promising alternative biopharmaceutical to treat growth hormone (GH) deficiency in children, and it was developed using a variety of technologies by several pharmaceutical companies. Most LAGH preparations, such as Fc fusion protein, are currently undergoing preclinical study and clinical trials. Accurate determination of bioactivity is critical for the efficacy of quality control systems of LAGH. The current in vivo rat weight gain assays used to determine the bioactivity of recombinant human GH (rhGH) in pharmacopoeias are time-consuming, expensive, and imprecise, and there are no recommended bioassays for LAGH bioactivity in pharmacopoeias. Therefore, we developed a cell-based bioassay for bioactivity determination of therapeutic long-acting Fc-fusion recombinant human growth hormone (rhGH-Fc) based on the luciferase reporter gene system, which is involved in the full-length human GH receptor (hGHR) and the SG (SIE and GAS) response element. The established bioassay was comprehensively validated according to the International Council for Harmonization (ICH) Q2 (R1) guidelines and the Chinese Pharmacopoeia, and is highly precise, time-saving, simple, and robust. The validated bioassay could be qualified for bioactivity determination during the research, development, and manufacture of rhGH-Fc, and other LAGH formulations.

A combined pore blockage and cake filtration model was applied to the virus filtration of an Fc-fusion protein using the three commercially available filters, F-1, F-2, and F-3 in a range of buffer conditions including sodium-phosphate and tris-acetate buffers with and without 200 mM NaCl at pH 7.5. The fouling behaviors of the three filters for the feed solutions spiked with minute virus of mice were described well by this combined model for all the solution conditions. This suggests that fouling of the virus filters is dominated by the pore blockage mechanism during the initial stage of the filtration and transformed to the cake filtration mechanism during the later stage of the filtration. Both flux and transmembrane resistance can be described well by this model. The pore blockage rate and the rate of increase of protein layer resistance over blocked pores are found to be affected by membrane properties as well as the solution conditions resulting from the modulation of interactions between virus, protein, and membrane by the solution conditions.

TPS3147 Background: Productive antitumor immune responses in nonclinical models depend on a type of dendritic cell (DC), conventional DC subtype 1 (cDC1), which in the context of cancer, primes tumor-reactive T cells through presentation of tumor-derived antigens. FMS-related tyrosine kinase 3 ligand (FLT3L) is a hematopoietic growth factor that binds to and activates FLT3 on terminally differentiated DCs. Activated FLT3 promotes proliferation, inhibits cell death, and is required for the differentiation, expansion, and maintenance of DCs in peripheral and lymphoid organs. GS-3583 is a fusion protein composed of the extracellular domain of recombinant human FLT3L fused to an engineered fragment crystallizable (Fc) region of human immunoglobulin G4. GS-3583 has PK properties that support sustained cDC in patients and potential combination with established immunotherapies. This phase 1b, open-label, multicenter, dose-finding study will evaluate safety, tolerability, PK, and preliminary efficacy of GS-3583 monotherapy in patients with advanced solid tumors (NCT04747470). Methods: Approximately 33 adults aged ≥18 years with a histologically or cytologically confirmed locally advanced or metastatic malignant solid tumor that is refractory to or intolerant of standard therapy or for which no standard therapy is available will be enrolled. The study employs a 3+3 dose escalation design in which GS-3583 is administered intravenously for up to 52 weeks or until progressive disease or unacceptable toxicity. Up to five dose escalation cohorts have been planned. The maximum tolerated dose is the highest dose with incidence of DLT in < 33% of 6 or more patients in the first 28 days of GS-3583 dosing; recommended phase 2 dose will be determined. Assessments include safety, PK, pharmacodynamics including cDCs, immunogenicity, and efficacy by RECIST 1.1 in CT/MRI imaging conducted every 8 weeks. Accrual at approximately 3-4 centers in the US is ongoing. Clinical trial information: NCT04747470.

5566 Background: AVB-500 is a first-in-class Fc fusion protein that binds the GAS6 ligand thereby inhibiting AXL signaling. Both GAS6 and AXL are highly expressed in high-grade serous ovarian cancer (HSGOC). This study evaluated safety, tolerability, and preliminary efficacy of AVB-500 in combination with pegylated liposomal doxorubicin (PLD) and paclitaxel (Pac) and determine the recommended Phase 2 dose (RP2D). Methods: Patients were enrolled in escalating dose cohorts of AVB-500 10mg/kg to 20mg/kg q2 weeks in combination with weekly Pac 80mg/m2 D1, 8, 15 q28 days or PLD 40mg/m2 D1 q28 days and assess for safety, pharmacokinetics, pharmacodynamics, and response by investigator, via RECIST v1.1. Results: A total of 53 patients with platinum-resistant HGSOC (PROC) were enrolled. A total of 23 patients received Pac + AVB-500 and 30 patients received PLD + AVB-500. Grade 3 or 4 treatment-related adverse events were observed in 4/23 (17%) and 2/30 (7%) PAC and PLD, respectively. No patients discontinued therapy due to an adverse event. Most events were related to known chemotherapy side effects. RP2D was identified as 15mg/kg. Confirmed overall response rate (ORR) with Pac+AVB-500 was 35% (8/23) including 2 CRs and 11% (3/28) in the PLD+AVB-500 subgroup. ORR was 19% (3/16) in patients with platinum free interval (PFI) of < 3 months versus (vs) 23% (8/35) in patients with PFI of 3-6 months. ORR was 11% (2/18) in patients with 1 prior treatment vs 27% (9/33) in patients with 2-3 prior lines of therapy. ORR in patients without prior bevacizumab was 33% (9/27) vs 8% (2/24) in those with prior bevacizumab. Patients treated with Pac combination and whose AVB-500 trough levels were above the minimal efficacious concentration (MEC) of 13.8mg/L achieved the greatest benefit with ORR, median PFS, and median OS of 43% (6/14), 3.9 months, and 17.8 months vs 22% (2/9), 2.8 months, and 8.7 months observed in those whose trough was below the MEC. Among the Pac treated subgroup, the ORR was 47% (13% CR) vs 0% for those with sAXL/GAS6 ratios > 0.773 compared to ratios 0.773. Conclusions: AVB-500 is a novel Fc fusion protein that binds the GAS6 ligand and inhibits AXL signaling. AVB-500 was well-tolerated in combination with Pac or PLD. This Ph1b trial suggested a higher ORR in the Pac treated subgroup, with C1D15 trough levels > 13.8mg/L (most consistently achieved at the 15mg/kg dose level). Exploratory analyses suggested that improved ORR may be observed in patients who have not been exposed to bevacizumab. The serum sAXL/GAS6 ratio may be a potential biomarker of pathway activation and identify patients who most benefit from Pac+AVB-500. The ORR in patients with PFI < 3 months or who had > 1 line of prior therapy were similar to those with 3-6 months PFI or ≤1 lines of therapy. Further development of AVB-500 15 mg/kg q2 weeks in combination with Pac is warranted in PROC. Clinical trial information: NCT03639246.

2559 Background: Conventional dendritic cells subtype 1 (cDC1) play a vital role in the priming and expansion of tumor specific CD8+ T cells and their recruitment to tumor microenvironment (TME). However, cDC1s are often underrepresented in the TME. Systemic administration of Fms-like tyrosine kinase 3 ligand (FLT3L), a hematopoietic growth factor that binds to FLT3 on myeloid and lymphoid progenitor cells, leads to expansion of cDC1s in the periphery which can then be recruited into the TME. We hypothesize that FLT3 pathway stimulation using GS-3583, a FLT3 agonist Fc fusion protein, has the potential to promote T cell mediated anti-tumor activity. Methods: This was a first-in-human placebo-controlled study of GS-3583 in healthy volunteers to evaluate the safety, PK, and PD of escalating single doses (ranging from 75μg to 2000μg) of GS-3583. The study was blinded to the subjects and the investigator. Each dose cohort enrolled 8-12 healthy subjects who received GS-3583 or placebo as single IV infusion at 3:1 ratio. Subjects were observed in the phase 1 unit for 15 days and then for 12 weeks as outpatients. As part of the PD evaluation, we investigated the changes in the number of cDC1s and cDC subtype 2 (cDC2) cells. Results: As of 8th Feb 2021, selected safety, PK and PD data from the first 3 cohorts were available. GS-3583 was well tolerated and all subjects had been discharged. To date, there have been no serious or grade 3 or higher adverse events. Preliminary PK analysis suggested dose-dependent increase in GS-3583 exposure (AUC and Cmax). Preliminary PD analysis shows that administration of GS-3583 resulted in dose-dependent increases in cDC1/cDC2 cells that peaked at day 5 or day 8 and returned to baseline within three weeks of drug administration. Conclusions: GS-3583 was safe and well tolerated and induced dose dependent expansion of dendritic cells in the periphery. In patients with cancer, this increase in dendritic cells can be utilized to enhance anti-tumor responses to immuno-oncology therapies.[Table: see text]

A newly developed hybrid Fc (hyFc) is a non-immunogenic and non-cytolytic Fc with intact Ig structure derived from human IgD and IgG4. It is fused with the human growth hormone (GXD-9) and was evaluated by various biophysical techniques. Two thermal transitions were evident by DSC, reflecting the unfolding of IgG4 and the conjugated protein. The highest Tm of the initial GXD-9 was 68.17°C and the Tm of the two domains were around 66°C and 70°C. Although Tm increased with decreasing concentration, which reflects increasing conformational stability, aggregation issues were still observed by DLS. This might be caused by decreasing or low zeta potential due to a highly complex structure. The protein was dialyzed to various pH (6.2-8.2) values to enhance conformational stability and to overcome aggregation issues. The results of CD spectroscopy were correlated with DSC measurements to evaluate its conformational stability. Changes in secondary structural contents were similar as determined by DSC and DLS. In conclusion, GXD-9 was found to be most stable at pH 7.0. The investigation of the biophysical stability of a hyFc-fusion protein has demonstrated a positive feasibility of developing more stable formulations to facilitate the initial drug development process for further clinical trials.

Introduction In haemophilia, prophylactic infusion of replacement factor can result in improvements in health-related quality of life (HRQoL) when compared with episodic treatment. The Haemophilia-specific Quality of Life (Haem-A-QoL) questionnaire assessed HRQoL in adults with severe haemophilia A or B who received prophylactic or episodic treatment with recombinant factor VIII or IX Fc fusion protein (rFVIIIFc or rFIXFc) in the A-LONG or B-LONG clinical studies. Aims Understand changes in HRQoL during the A-LONG and B-LONG trials. Methods Group-level and individual-level changes over time for the Haem-A-QoL key domains of ‘Physical Health’ and ‘Sports & Leisure,’ and ‘Total Score’ were evaluated in adults through baseline and 6-month HRQoL assessments. Previously determined responder definitions (RDs) were used for evaluating meaningful subject-level HRQoL improvements. Results The analysis included 67 A-LONG and 51 B-LONG subjects who completed the Haem-A-QoL (baseline and 6 months). While HRQoL improvements were observed among all treatment groups, greater improvements in HRQoL were observed among subjects who received episodic treatment pre-study (and prophylaxis on-study) compared to those who received hyphenate prophylaxis. Applying the RDs for interpreting 6-month changes, 47.4%/33.3% (‘Physical Health’), 35.9%/50.0% (‘Sports & Leisure’) and 23.9%/33.3% (‘Total Score’) of A-LONG subjects who received individualized or weekly prophylaxis were classified as HRQoL responders. In B-LONG, 69.2%/57.9% (‘Physical Health’), 44.4%/56.7% (‘Sports & Leisure’) and 41.7%/44.1% (‘Total Score’) of subjects who received individualized or weekly prophylaxis were classified as HRQoL responders. Conclusion Changes in Haem-A-QoL key domains and ‘Total Score’ suggest that prophylaxis with long-acting rFVIIIFc or rFIXFc resulted in meaningful HRQoL improvements.

The T-cell response is a critical component of controlling many cancers and viral diseases and recently developed T-cell based therapies have become very popular and effective treatment methods. Adoptive cell therapy involves the isolation of a patient’s or donor’s T-cells, expansion or modification ex vivo, and then administration into the patient. The recent FDA approval of an adoptive cell therapy involving an engineering antigen receptor has solidified this technique from a passing fad to a legitimate and effective technique for treating cancer. T-cells are able to recognize a vast range of target antigens through the use of a T-cell receptor (TCR) which targets small peptide fragments presented on the surface of diseased cells in the form of major histocompatibility complexes (pMHC). Further improvement of these therapies and methods requires a deeper understanding of how these TCR-pMHC interactions relate to functionality. Cytomegalovirus is a persistent viral infection that can reactivate periodically during periods of immunodepression. Viral control is mediated by a strong T-cell response and it can cause life-threatening complications in immunosuppressed individuals.Additionally, the T-cell response after chronic CMV reactivation becomes focused to a few individual clonotypes bearing ‘public’ TCRs that have shared sequences between individuals and are often high affinity. This makes CMV an excellent model for studying TCR-pMHC interactions. Several attempts to improve functionality of natural TCRs by engineering for higher affinity have resulted loss of specificity or reduced activation. Methods for direct selection of TCRs with improved activity are currently being developed, however they are very limited, whereas there are a variety of methods for affinity maturation of TCRs. A firm understanding of how TCR-pMHC binding affinity relates to activation would greatly benefit all T cell therapies. This work attempts to provide a better understanding of the relationship between TCR-pMHC affinity and T-cell activity by characterizing TCRs engineered in multiple platforms to obtain a range of affinities while maintaining antigen specificity. Additionally, we observe the relationship between affinity and activity of in vivo derived TCRs against CMV to help aid adoptive cell therapies, as well as the development of a vaccine to elicit potent T-cell responses.

INTRODUCTION: Variability in FVIII measurement is a recognized problem. There are limited data for samples containing recombinant Factor VIII Fc fusion protein (rFVIIIFc). Many studies use samples for which factor concentrate has been spiked into FVIII deficient plasma in vitro. This approach requires validation. AIM/METHODS: Four samples were distributed in a UK National External Quality Assessment Scheme for Blood Coagulation (NEQAS BC) survey. One contained Advate (full-length recombinant FVIII) (rFVIII) added to FVIII deficient plasma, one was from a severe haemophilia A patient after infusion of Advate, one was prepared by addition of rFVIIIFc (marketed as Elocta/Eloctate) to FVIII deficient plasma and the fourth was collected from a severe haemophilia A patient following rFVIIIFc (Eloctate) infusion. Fifty-three haemophilia centres (UK and Scandinavia) performed one-stage FVIII assays and 27 performed chromogenic FVIII assays. RESULTS/CONCLUSIONS: One-stage assays gave significantly lower results than chromogenic assays by 7% (P

Introduction: Recent haemophilia treatment advances include new recombinant FVIII (rFVIII) products with improved pharmacokinetic (PK) properties that aim to reduce the burden of prophylaxis. These treatments are commonly referred to as extended half-life rFVIII products (EHL rFVIII). There is no uniform definition of what constitutes an EHL rFVIII. Such a definition would help physicians, patients and funders understand the properties of standard and EHL rFVIIIs and thus provide clarity when selecting an EHL in clinical settings. Aim: To critically assess the published evidence on new and emerging rFVIII products in order to propose a definition to classify EHL rFVIIIs. Methods: We systematically searched PubMed, EMBASE and regulatory authorities (FDA/EMA/Health Canada) websites for publications and regulatory submissions describing prospective crossover PK studies evaluating rFVIIIs that demonstrate improved PK parameters in adults and adolescents with severe haemophilia A. Results: Following critical analyses of the published data, we developed a holistic approach to defining rFVIIIs as EHLs, which requires all of the following: (i) using technology designed to extend rFVIII half-life; (ii) lacking bioequivalence with a standard rFVIII comparator-above the FDA/EMA cut-off of 125% for the 90% confidence intervals for area under the curve ratio; and (iii) having an extended half-life ratio measured in a PK comparator crossover study. Conclusion: In this systematic review, a pragmatic definition of EHL rFVIII has been proposed that should provide better clarity in clinical discussions surrounding the appropriate use of rFVIII products. At present, only products using PEGylation or Fc fusion half-life extension technology meet the proposed criteria for definition of EHL rFVIII. (Less)

Objective Hybrid Fc-fused rhGH (GX-H9) is a long-acting recombinant human growth hormone (GH) under clinical development for both adults and children with GH deficiency (GHD). We compared the safety, pharmacokinetics and pharmacodynamics of weekly and every other week (EOW) dosages of GX-H9 with those of daily GH administration in adult GHD (AGHD) patients. Design This was a randomized, open-label, active-controlled and dose-escalation study conducted in 16 endocrinology centers in Europe and Korea. Methods Forty-five AGHD patients with or without prior GH treatment were enrolled. Patients with prior GH treatments were required to have received the last GH administration at least 1 month prior to randomization. Subjects were sequentially assigned to treatment groups. Fifteen subjects were enrolled to each treatment group and randomly assigned to receive either GX-H9 or Genotropin (4:1 ratio). GX-H9 dosage regimens for Groups 1, 2 and 3 were 0.1 mg/kg weekly, 0.3 mg/kg EOW and 0.2 mg/kg EOW, respectively. All Genotropin-assigned subjects received 6 µg/kg Genotropin, regardless of treatment group. Main outcome analyses included measurements of serum insulin-like growth factor 1 (IGF-I), safety, pharmacokinetics, pharmacodynamics and immunogenicity. Results Mean GX-H9 peak and total exposure increased with an increase in dose after a single-dose administration. The mean IGF-I response was sustained above baseline over the intended dose interval of 168 h for the weekly and 336 h for the EOW GX-H9 groups. Safety profiles and immunogenicity were not different across the treatment groups and with Genotropin. Conclusions GX-H9 has the potential for up to twice-monthly administration.

Introduction Recombinant factor VIII Fc fusion protein (rFVIIIFc) is a novel recombinant factor VIII with a prolonged half-life, developed for the treatment of hemophilia A. Studies that evaluated the toxicological effects of rFVIIIFc in 2 pharmacologically relevant species, cynomolgus monkeys and Sprague Dawley rats, are reported here. Materials and Methods In repeat-dose toxicology studies, rats and monkeys received 0, 50, 250, or 1000 IU/kg rFVIIIFc every other day for 4 weeks. In a high-dose tolerance study, monkeys received 1 rFVIIIFc dose of 3000, 10,000, or 20,000 IU/kg. Evaluations included in-life observations, laboratory and post-mortem evaluations, pharmacokinetics, and local tolerance. Allometric scaling, using data from 4 animal species and humans, was used to evaluate the relationship between animal and human pharmacokinetics. Results rFVIIIFc was well tolerated with no adverse toxicological findings directly attributable to rFVIIIFc. As expected, antibodies to this fully human protein developed in rats and monkeys in a time-dependent fashion following repeated dosing, leading to increased clearance in both species. There were no local reactions (infusion site) or evidence of thrombosis at high doses in rats and monkeys. Allometric scaling demonstrated more rapid clearance in small animals compared with humans and a volume of distribution (steady state) proportional to body weight across species, suggesting that animal pharmacokinetics are predictive of human pharmacokinetics. Conclusions Repeated doses of rFVIIIFc in 2 relevant animal species and high doses of rFVIIIFc in monkeys were well tolerated. These results supported the clinical safety of rFVIIIFc observed in phase 1/2a and phase 3 clinical trials.

Recycling of antibodies and Fc containing therapeutic proteins by the neonatal Fc receptor (FcRn) is known to prolong their persistence in the bloodstream. Fusion of Fc fragment of IgG1 to other proteins is one of the strategies to improve their pharmacokinetic properties. Accurate measurement of Fc-FcRn binding provides information about the strength of this interaction, which in most cases correlates with serum half-life of the protein. It can also offer insight into functional integrity of Fc region. We investigated FcRn binding activity of a large set of Fc-fusion samples after thermal stress by the method based on AlphaScreen technology. An unexpected significant increase in FcR binding was found to correlate with formation of aggregates in these samples. Monomer purified from a thermally-stressed sample had normal FcRn binding, confirming that its Fc portion was intact. Experiments with aggregates spiked into a sample with low initial aggregation level, demonstrated strong correlation between the level of aggregates and FcRn binding. This correlation varied significantly in different methods. By introducing modifications to the assay format we were able to minimize the effects of aggregated species on FcRn binding, which should prevent masking functional changes of Fc-fusion protein. Biolayer interferometry (BLI) was used as an alternative method to measure FcRn binding. Both optimized AlphaScreen- and BLI-based assays were sensitive to structural changes in Fc portion of the molecule, such as oxidation of methionines 252 and 428, and therefore suitable for characterization of FcRn binding.

Introduction Recombinant factor IX Fc fusion protein (rFIXFc) is a recombinant coagulation factor composed of a single molecule of recombinant factor IX (rFIX) covalently fused to the Fc domain of human immunoglobulin G1 (IgG1) with no intervening sequence. An extensive nonclinical program was performed to support the clinical development of rFIXFc for treatment of people with hemophilia B. Materials and Methods Repeat-dose toxicology studies of rFIXFc were performed in 2 relevant species: Sprague Dawley rats (4-week study) and cynomolgus monkeys (5- and 27-week studies). Assessments included in-life observations, electrocardiograms (monkeys only), laboratory evaluations (including hematology and blood chemistry), postmortem analyses, local tolerance, and pharmacokinetics (PK). Allometric scaling was performed with PK data from multiple species, including humans. Local tolerance (single-dose study) and thrombogenic potential (Wessler stasis model) of rFIXFc were tested in New Zealand White rabbits. Results There were no significant local or systemic toxicity findings in the repeat-dose studies. Allometric scaling data suggested that animal rFIXFc PK results are predictive of human PK parameters. There were no findings from the local tolerance study in rabbits; thrombogenic activity was less than that elicited by rFIX and a prothrombin complex concentrate, and similar to vehicle control. Conclusions rFIXFc was well tolerated in toxicology studies and demonstrated a low thrombogenic potential. These results are consistent with phase 1/2a and phase 3 clinical studies of rFIXFc in people with hemophilia B.

Introduction The safety, efficacy and prolonged half-life of recombinant factor VIII Fc fusion protein (rFVIIIFc) in previously treated patients with severe haemophilia A was demonstrated in the phase 3 A-LONG and Kids A-LONG studies. Here, we report interim safety and efficacy data from the rFVIIIFc extension study, ASPIRE (ClinicalTrials.gov #NCT01454739). Methods Eligible subjects could enrol in ASPIRE upon completing A-LONG or Kids A-LONG. There were four treatment groups: individualized prophylaxis; weekly prophylaxis; modified prophylaxis (for subjects in whom optimal treatment could not be achieved with individualized or weekly prophylaxis); and episodic treatment. The primary endpoint was development of inhibitors. Results A total of 150 A-LONG subjects and 61 Kids A-LONG subjects enrolled in ASPIRE. As of the interim data cut (6 January 2014), the median time on study was 80.9 (A-LONG) and 23.9 (Kids A-LONG) weeks. The majority of subjects (A-LONG, 92.0%; Kids A-LONG, 57.4%) had ≥100 cumulative rFVIIIFc exposure days. No inhibitors were observed. Adverse events were generally consistent with those expected in the general haemophilia A population. Median annualized bleeding rates (ABRs) were low with individualized [A-LONG: 0.66; Kids A-LONG: 0.00 (

Prophylactic infusion of factor VIII (FVIII) prevents joint bleeding and other hemorrhages in patients with hemophilia A. Conventional FVIII concentrates have a short half-life, with an average of about 12 h in adults, ranging in individual patients between 6 and 24 h, and even shorter in younger children. Therefore, effective prophylaxis requires frequent intravenous injection, usually three times per week or every other day. Several technologies are currently under investigation to extend the half-life of FVIII, including Fc fusion (Eloctate, Elocta, efmoroctocog alfa), addition of polyethylene glycol (turoctocog alfa pegol [N8-GP], BAY 94-9027, BAX 855), and a single-chain construct (CSL627). This review summarizes characteristics of products in clinical development and discusses their potential benefits.

Stefan R. Schmidt consolidates the hugely diverse field of fusion proteins and their application in the creation of biopharmaceuticals. The text is replete with case studies and clinical data that inform and intrigue the reader as to the myriad possibilities available when considering the creation of a fusion protein. This valuable text will serve the novice as a broad introduction or the seasoned professional as a thorough review of the state of the art. The first marketed therapeutic recombinant protein was human insulin (Humulin® R). Its approval in 1982 was followed by other such products, including erythropoietin (EPO), interferon (IFN), and tissue plasminogen activator (tPa). Since the 1980s, the number and general availability of recombinant products that replace natural proteins harvested from animal or human sources has increased considerably. Following the initial success, researchers started de novo designs of therapeutic proteins that do not occur in nature. The first of these new drugs to be ...

To develop a rapid process analytical technology (PAT) tool that can measure sialic acid content of an Fc-fusion protein from cell culture samples.A statistical significant correlation between the sialic acid content and size-exclusion chromatography (SEC)-HPLC retention time of an Fc-fusion protein was observed when analyzing the titer of the samples. Using linear fitting analysis, the data fit the model well with R (2) = 0.985. Based on the SDS-PAGE and oligosaccharide analysis, we speculate that the amounts of the glycans could expand the structure of the Fc-fusion protein. This was manifested by the SEC-HPLC method in which proteins were separated based on its molecular size. In order to development a robust PAT method, an internal standard was used to improve the precision of the method by reducing systematic errors. We found the change of SEC retention time (delta t) and sialic acid content were highly correlated (R (2) = 0.992). This method was further validated by a 1500 l production process.SEC-HPLC is a promising PAT tool to monitor the sialic acid content of Fc-fusion protein during biomanufacturing or medium optimization processes.

A robust, economical process should leverage proven technology, yet be flexible enough to adopt emerging technologies which show significant benefit. Antibody and Fc-fusion processes may capitalize on the high selectivity of an affinity capture step by reducing the total number of chromatographic steps to 2. Risk associated with this approach stems from the potentially increased time frame needed for process development as well as unforeseen changes in impurity profile during first scale-up of drug substance (DS) for animal toxicology and clinical phase I trials (FIH) production, which could challenge a two-step process to the point of failure. Two different purification strategies were pursued during process development for an FIH process of a dAB-Fc fusion protein. A two-step process was compared to a three-step process. The two-step process leveraged additives to maximize impurity reduction during affinity capture. While wash additives in combination with a mixed mode chromatography met all impurity reduction requirements, HCP levels were still higher as compared to the three-step process. The three-step process was implemented for manufacture of clinical material to mitigate risk.

构建了人肿瘤坏死因子受体II胞外区、人脂联素球部与人IgG1 Fc的融合基因sTNFRII-gAD-Fc的真核表达载体，在CHO-S细胞中快速高效表达，探索了无血清悬浮流加培养工艺。应用重组PCR将人IgG1 Fc基因片段与sTNFRII-gAD基因片段融合，构建pMH3-sTNFRII-gAD-Fc表达载体，电转染至CHO-S细胞，挑选G418抗性的高表达单克隆，斑点杂交半定量分析和Western blot分析sTNFRII-gAD-Fc的表达，Protein A-Agarose纯化，以及拮抗TNFα的生物活性测定。最后在摇瓶、转瓶及生物反应器中进行了逐级放大的无血清悬浮批次及流加培养，即：2.0 × 106 cells/mL的接种密度，当达到4.0 × 106 cells/mL以上时开始流加培养并以每日葡萄糖的残余量2 g/L左右作为流加体积的控制参数。成功构建pMH3- sTNFRII-gAD-Fc表达载体，检测到sTNFRII-gAD-Fc在CHO-S细胞培养上清中以二聚体和多聚体形式表达，获得了高表达(75 μg/mL)单克隆细胞株，且该蛋白具有显著抑制TNFα杀伤L929细胞的活性。在摇瓶中无血清批次培养和转瓶及生物反应器中流加培养时产量分别为10.0 mg/L、18.3 mg/L和20.5 mg/L。利用CHO-S细胞系统成功实现了sTNFRII-gAD-Fc 融合蛋白的快速高效表达，为建立一套高密度、高效表达该融合蛋白的悬浮流加培养中试工艺打下了良好基础。 In order to produce the soluble TNF receptor (sTNFR) II with good neutralizing activity against TNFα, we constructed the fusion gene sTNFRII-gAD-Fc, which encoded human sTNFRII, the globular domain of adiponectin (gAD) and IgG1 Fc, and efficiently expressed it through a “GC-rich” method for mammalian gene expression in CHO-S cells, and further established the process of serum-free suspension fed-batch culture. The fusion gene of sTNFRII-gAD-Fc was obtained by recombinant PCR, and then cloned into pMH3 expression plasmid with GC-rich motif. The plasmid was electrotransfected into CHO-S cells, which were selected by G418. sTNFRII-gAD-Fc fusion protein was analyzed by dot blot and Western blot. The TNFα-antagonizing activity of sTNFRII-gAD- Fc was determined through TNFα-induced L929 cytotoxicity assay. The suspension cultures of sTNFRII-gAD-Fc-expressing CHO-S cells were performed in a step-wise manner. We assessed the expression levels of sTNFRII-gAD-Fc in batch culture in shake flaskes (500 mL), fed-batch culture in roller bottles (2 L), and the bioreactor (7.5 L) with inoculating concentration of 2 × 106 cells/ml of sTNFRII-gAD-Fc-expressing CHO-S cells, respectively. Having reached up to more than 4 × 106 cells/ml in fed-batch cultures, the cells were added semi-continuously with the feed medium to keep the glucose concentration at 2 g/L. Stable expression clones (75 μg/mL) were obtained, and sTNFRII-gAD-Fc fusion protein was expressed as dimer and polymer forms in the supernatant, and displayed very strong TNFα-neutralizing activity. The yields of sTNFRII-gAD-Fc fusion protein were about 10.0 mg/L, 18.3 mg/L and 20.5 mg/L, respectively, in 60 mL batch culture, 200 mL and 3 L fed-batch cultures. Our efficient expression of sTNFRII-gAD-Fc fusion protein by CHO-S cells had laid a good basis for the development of pilot production process in the suspension fed-batch culture.

SummaryThe use of plasma-derived and recombinant coagulation factors for the treatment of haemophilia A and B is well established and permits patients to live a relatively normal life. In order to improve treatment options, several products are in development, which have a prolonged duration of action, thus enabling less frequent prophylactic dosing and aiming to reduce the burden of treatment. Several innovative approaches are being pursued to extend the half-life of factor VIIa, factor VIII and factor IX, utilising technologies such as Fc fusion, recombinant albumin fusion and addition of polyethyleneglycol (PEG) (PEG ylation). These methods prolong the time in the circulation by reducing degradation and elimination. This review summarises the technologies and products in development and their stages of development, and also discusses their pros and cons.

Erythropoietin Fc (EPO-Fc) fusion proteins are potential drug candidates that have been designed for the treatment of anemia in humans by stimulating erythrocyte production. Such compounds can be considered performance-enhancing agents that may be used by athletes in endurance sports. This study describes the primary structure of commercially available EPO-Fc based on comprehensive liquid chromatography coupled with mass spectrometry (LC–MS) analysis. A bottom-up approach and the intact molecular weight (MW) measurement of deglycosylated protein and its IdeS proteolytic fractions was used to determine the amino acid sequence of EPO-Fc. Using multiple proteases, peptides covering unknown fusion breakpoints (spacer peptides) were identified. We demonstrated that “spacer peptides” can be used in the determination of EPO-Fc fusion proteins in biological samples using common LC–tandem MS methods.

Monoclonal antibodies (mAbs) and fusion proteins with an Fc portion of immunoglobulin G (IgG) are emblematic of the remarkable expansion of biopharmaceuticals. Despite their biological origin, these products display an interindividual variability in their efficacy and/or side effects, which must be taken into consideration. Biological monitoring allowing for adapted prescription and dose adjustments may lead to therapeutic optimization and limitation of the high costs of these drugs. Herein, we review the biological theranostic of mAbs and Fc fusion proteins, including pre-treatment analyses, monitoring of efficacy, therapeutic drug monitoring, and monitoring of side effects. Supported by concrete evidence, a specific interest is given to individualised therapeutic monitoring that combines intention to treat, biomarkers of efficacy and adaptation of serum concentrations.

The development of a new generation of coagulation factors with improved pharmacokinetic profile will change the paradigm of treatment of persons with hemophilia (PWH). The standard treatment in PWH is represented by regular long-term prophylaxis that, given intravenously twice or thrice weekly, is associated with a not-negligible burden on patients’ quality of life. The availability of drugs with improved pharmacokinetic profile may improve prophylaxis feasibility and protection against bleeding episodes. This article summarizes the main results obtained from clinical trials with modified factor VIII (FVIII) and factor IX (FIX) molecules. Published literature on new molecules for replacement treatment in hemophilia A and B was retrieved using PubMed search, and all ongoing clinical trials have been researched via www.clinicaltrials.gov. Such new molecules are usually engineered to have a longer plasma half-life than that which has been obtained by chemical modification (i.e., conjugation with polyethylene glycol, PEG) or by creating recombinant fusion proteins. Results from phase I/III studies in previously treated adults and children are now available for the vast majority of new products, including the results of their use in a surgical setting. On the contrary, trials involving previously untreated patients are still ongoing for all and results not yet available.

Introduction: Current prophylactic management of hemophilia A (HemA) involves administration of intravenous factor VIII (FVIII) 3 – 4 times weekly. Fc fusion protein technology uses the natural neonatal Fc receptor (FcRn) receptor to extend the half-life of Fc-associated therapeutic proteins. A recombinant factor VIII Fc fusion protein (rFVIIIFc) with a half-life 1.5 – 1.7-fold longer than unmodified rFVIII has now been developed. Areas covered: This review outlines the FcRn recycling pathway and the development of Fc fusion drugs. rFVIIIFc was successfully tested in HemA mice and dogs and demonstrated a good safety profile in a Phase I/IIa study in previously treated subjects with severe hemophilia A. In a pivotal Phase III study of subjects with severe hemophilia A, rFVIIIFc had a prolonged half-life compared with rFVIII and was well tolerated and efficacious in the prevention and treatment of bleeding events. Expert opinion: A shift from alternate day dosing to once or twice weekly dosing for HemA pati...

Hemophilia B is a genetic disease caused by mutation of the gene for coagulation protein Factor IX. When severe, the disease leads to spontaneous life-threatening bleeding episodes. Current therapy requires frequent intravenous infusions of therapeutic recombinant or plasma-derived protein concentrates containing Factor IX. Alprolix™ (recombinant Factor IX Fc fusion protein), is a therapeutic Factor IX preparation that has been engineered for a prolonged half-life in circulation, has completed pivotal clinical trials and has been approved recently in the USA, Canada, Australia and Japan for use in the clinic for patients with hemophilia B. This promising therapy should allow patients to use fewer infusions to maintain appropriate Factor IX activity levels in all clinical settings, and its use may be indicated in both on demand and prophylactic treatments.

Ligand-receptor interactions represent essential biological triggers which regulate many diverse and important cellular processes. We have developed a discovery-based proteomic biochemical protocol which couples affinity purification with multidimensional liquid chromatographic tandem mass spectrometry (LCLC-MS/MS) and bioinformatic analysis. Compared to previous approaches, our analysis increases sensitivity, shortens analysis duration, and boosts comprehensiveness. In this protocol, receptor extracellular domains are fused with the Fc region of IgG to generate fusion proteins that are purified from transfected HEK293T cells. These “ecto-Fcs” are coupled to protein A beads and serve as baits for binding assays with prey proteins extracted from rodent brain. After capture, the affinity purified proteins are digested into peptides and comprehensively analyzed by LCLC-MS/MS with ion trap mass spectrometers. In four working days, this protocol can generate shortlists of candidate ligand-receptor protein-protein interactions. Our “Ecto-Fc MS” approach outperforms antibody-based approaches and provides a reproducible and robust framework to identify extracellular ligand – receptor interactions.

Hemophilia B is a rare congenital bleeding disorder characterized by a deficiency of coagulation factor IX (FIX). Hemophilia B patients experience mild-to-severe bleeding complications according to the degree of FIX defect. Prophylaxis, with regular infusion of FIX concentrates, is nowadays, the mainstay of hemophilia care. However, because the relatively short half-life of such products necessitates frequent infusions and thus makes patients' adherence difficult, a number of strategies have been implemented to improve the pharmacokinetics of FIX clotting factors.This review summarizes the main results of Phase I/II and III studies on new FIX molecules engineered to have a longer half-life. Several technologies are being applied to extend FIX half-life, including Fc fusion, recombinant (r) albumin fusion and the addition of PEG polymers.By prolonging the FIX half-life up to 5 times, long-acting FIX products are expected to substantially improve the management of hemophilia B patients, allowing less frequent infusions and improving patients' adherence to prophylactic regimens and individualized treatments. Some of them are at an advanced stage of development, such as the rFIX-Fc which has been launched in March 2014. Along with the ongoing Phase III trials, long-term post-marketing surveillance studies are needed to assess their safety and effectiveness and their impact on patients' quality of life.

The goal of drug delivery is to improve the safety and therapeutic efficacy of drugs. This review focuses on delivery platforms that are either derived from endogenous pathways, long-circulating biomolecules and cells or that piggyback onto long-circulating biomolecules and cells. The first class of such platforms is protein-based delivery systems—albumin, transferrin, and fusion to the Fc domain of antibodies—that have a long-circulation half-life and are designed to transport different molecules. The second class is lipid-based delivery systems—lipoproteins and exosomes—that are naturally occurring circulating lipid particles. The third class is cell-based delivery systems—erythrocytes, macrophages, and platelets—that have evolved, for reasons central to their function, to exhibit a long life-time in the body. The last class is small molecule-based delivery systems that include folic acid. This article reviews the biology of these systems, their application in drug delivery, and the promises and limitations of these endogenous systems for drug delivery. Biotechnol. Bioeng. 2014;111: 1699–1716. © 2014 Wiley Periodicals, Inc.