Multivariate data analysis of process parameters affecting the growth and productivity of stable Chinese hamster ovary cell pools expressing SARS-CoV-2 spike protein as vaccine antigen in early process development.

The recent COVID-19 pandemic revealed an urgent need to develop robust cell culture platforms which can react rapidly to respond to this kind of global health issue. Chinese hamster ovary (CHO) stable pools can be a vital alternative to quickly provide gram amounts of recombinant proteins required for early-phase clinical assays. In this study, we analyze early process development data of recombinant trimeric spike protein Cumate-inducible manufacturing platform utilizing CHO stable pool as a preferred production host across three different stirred-tank bioreactor scales (0.75, 1, and 10 L). The impact of cell passage number as an indicator of cell age, methionine sulfoximine (MSX) concentration as a selection pressure, and cell seeding density was investigated using stable pools expressing three variants of concern. Multivariate data analysis with principal component analysis and batch-wise unfolding technique was applied to evaluate the effect of critical process parameters on production variability and a random forest (RF) model was developed to forecast protein production. In order to further improve process understanding, the RF model was analyzed with Shapley value dependency plots so as to determine what ranges of variables were most associated with increased protein production. Increasing longevity, controlling lactate build-up, and altering pH deadband are considered promising approaches to improve overall culture outcomes. The results also demonstrated that these pools are in general stable expressing similar level of spike proteins up to cell passage 11 (~31 cell generations). This enables to expand enough cells required to seed large volume of 200-2000 L bioreactor.

A Biosensor Assay Based on Coiled-Coil-Mediated Human ACE2 Receptor Capture for the Analysis of Its Interactions with the SARS-CoV-2 Receptor Binding Domain.

Surface plasmon resonance (SPR)-based biosensing enables the characterization of protein-protein interactions. Several SPR-based approaches have been designed to evaluate the binding mechanism between the angiotensin-converting enzyme 2 (ACE2) receptor and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein leading to a large range of kinetic and thermodynamic constants. This chapter describes a robust SPR assay based on the K5/E5 coiled-coil capture strategy that reduces artifacts. In this method, ACE2 receptors were produced with an E5-tag and immobilized as ligands in the SPR assay. This chapter details methods for high-yield production and purification of the studied proteins, functionalization of the sensor chip, conduction of the SPR assay, and data analysis.

Influence of variant-specific mutations, temperature and pH on conformations of a large set of SARS-CoV-2 spike trimer vaccine antigen candidates.

SARS-CoV-2 subunit vaccines continue to be the focus of intense clinical development worldwide. Protein antigens in these vaccines most commonly consist of the spike ectodomain fused to a heterologous trimerization sequence, designed to mimic the compact, prefusion conformation of the spike on the virus surface. Since 2020, we have produced dozens of such constructs in CHO cells, consisting of spike variants with different mutations fused to different trimerization sequences. This set of constructs displayed notable conformational heterogeneity, with two distinct trimer species consistently detected by analytical size exclusion chromatography. A recent report showed that spike ectodomain fusion constructs can adopt an alternative trimer conformation consisting of loosely associated ectodomain protomers. Here, we applied multiple biophysical and immunological techniques to demonstrate that this alternative conformation is formed to a significant extent by several SARS-CoV-2 variant spike proteins. We have also examined the influence of temperature and pH, which can induce inter-conversion of the two forms. The substantial structural differences between these trimer types may impact their performance as vaccine antigens.

Preclinical evaluation of manufacturable SARS-CoV-2 spike virus-like particles produced in Chinese Hamster Ovary cells.

BACKGROUND: As the COVID-19 pandemic continues to evolve, novel vaccines need to be developed that are readily manufacturable and provide clinical efficacy against emerging SARS-CoV-2 variants. Virus-like particles (VLPs) presenting the spike antigen at their surface offer remarkable benefits over other vaccine antigen formats; however, current SARS-CoV-2 VLP vaccines candidates in clinical development suffer from challenges including low volumetric productivity, poor spike antigen density, expression platform-driven divergent protein glycosylation and complex upstream/downstream processing requirements. Despite their extensive use for therapeutic protein manufacturing and proven ability to produce enveloped VLPs, Chinese Hamster Ovary (CHO) cells are rarely used for the commercial production of VLP-based vaccines. METHODS: Using CHO cells, we aimed to produce VLPs displaying the full-length SARS-CoV-2 spike. Affinity chromatography was used to capture VLPs released in the culture medium from engineered CHO cells expressing spike. The structure, protein content, and glycosylation of spikes in VLPs were characterized by several biochemical and biophysical methods. In vivo, the generation of neutralizing antibodies and protection against SARS-CoV-2 infection was tested in mouse and hamster models. RESULTS: We demonstrate that spike overexpression in CHO cells is sufficient by itself to generate high VLP titers. These VLPs are evocative of the native virus but with at least three-fold higher spike density. In vivo, purified VLPs elicit strong humoral and cellular immunity at nanogram dose levels which grant protection against SARS-CoV-2 infection. CONCLUSIONS: Our results show that CHO cells are amenable to efficient manufacturing of high titers of a potently immunogenic spike protein-based VLP vaccine antigen.

Virus-like particles (VLPs) have a structure that is similar to viruses but they cannot cause infection or illness. If VLPs are injected into the body they produce an immune response similar to that seen following infection by a virus. This means that VLPs can be used as vaccines against viruses that cause illness in people. Many drugs, named biologics, are manufactured using living cells, including cells that were originally derived from Chinese Hamster Ovaries (CHO cells). We developed a simple method to produce VLPs similar to the SARS-CoV-2 virus in CHO cells. We show that vaccination of rodents with these VLPs prevents them from becoming ill following infection with SARS-CoV-2. These VLPs could become a part of an alternative, easily produced vaccine for the prevention of COVID-19 in humans.

Impact of the temperature on the interactions between common variants of the SARS-CoV-2 receptor binding domain and the human ACE2.

Several key mutations in the Spike protein receptor binding domain (RBD) have been identified to influence its affinity for the human Angiotensin-Converting Enzyme 2 (ACE2). Here, we perform a comparative study of the ACE2 binding to the wild type (Wuhan) RBD and some of its variants: Alpha B.1.1.7, Beta B.1.351, Delta B.1.617.2, Kappa B.1.617.1, B.1.1.7 + L452R and Omicron B.1.1.529. Using a coiled-coil mediated tethering approach of ACE2 in a novel surface plasmon resonance (SPR)-based assay, we measured interactions at different temperatures. Binding experiments at 10 °C enhanced the kinetic dissimilarities between the RBD variants and allowed a proper fit to a Langmuir 1:1 model with high accuracy and reproducibility, thus unraveling subtle differences within RBD mutants and ACE2 glycovariants. Our study emphasizes the importance of SPR-based assay parameters in the acquisition of biologically relevant data and offers a powerful tool to deepen our understanding of the role of the various RBD mutations in ACE2 interaction binding parameters.

Primary Care Practices in Western North Carolina: Adaptation to the COVID-19 Pandemic and Ongoing Challenges.

Introduction: The COVID-19 pandemic has created unique challenges for primary care practices while also highlighting their importance in the pandemic response. To understand primary care practice needs, a survey was conducted of practices in Western North Carolina. Methods: Phase 2 of a primary care needs assessment was administered to 63 practices in Western North Carolina over the course of six weeks, from July 23 to August 31, 2021. Results: Most practices were operating with normal hours, though some still operated with reduced hours. Many practices reported insufficient personal protective equipment (PPE) supplies. While most practices provided at least some care via telehealth, practices cited different barriers to providing telehealth, with patient technology challenges being the most frequently cited. Discussion: Practices have adapted to the restrictions of the pandemic, but many are still vulnerable, and the patients they serve may face reduced access to care due to practice limitations or barriers to telehealth. Practices play a critical role in providing care to patients throughout the pandemic and continue to assist in pandemic response by providing COVID-19 testing and other services. Conclusion: Primary care practices in Western North Carolina continue to provide care to patients and support the overall pandemic response. The pandemic has highlighted the need to include primary care in emergency response efforts. Ongoing work will allow North Carolina to reach practices more effectively in future crises via the newly created NC Responds system, which allows primary care practices to be contacted in the event of a public health emergency.

Foot-and-Mouth Disease: Optimization, Reproducibility, and Scalability of High-Yield Production of Virus-Like Particles for a Next-Generation Vaccine.

Inactivated Foot-and-Mouth Disease (FMD) vaccine has proven to be effective in the control of the disease. However, its production has some disadvantages, including the costly biosafety facilities required for the production of huge amounts of growing live virus, the need of an exhaustive purification process to eliminate non-structural proteins of the virus in the final formulations in order to differentiate infected from vaccinated animals and variable local regulatory restrictions to produce and commercialize the vaccine. Thus, a novel vaccine against FMD that overcome these restrictions is desirable. Although many developments have been made in this regard, most of them failed in terms of efficacy or when considering their transferability to the industry. We have previously reported the use of transient gene expression in mammalian cells to produce FMD virus-like particles (VLPs) as a novel vaccine for FMD and demonstrated the immunogenicity of the recombinant structures in animal models. Here, we report the optimization of the production system by assaying different DNA:polyethylenimine concentrations, cell densities, and direct and indirect protocols of transfection. Also, we evaluated the reproducibility and scalability of the technology to produce high yields of recombinant VLPs in a cost-effective and scalable system compatible with industrial tech-transfer of an effective and safe vaccine.

COVID-19 Needs Assessment and Support for Primary Care Practices in Western North Carolina.

Background: It is critical to ensure that Primary Care Providers (PCPs) have adequate personal protective equipment (PPE), supplies, training, staffing, and contingency planning during pandemics, particularly in rural areas. In March 2020, during the onset of the COVID-19 pandemic, the Mountain Area Health Education Center (MAHEC), in collaboration with the Cecil G. Sheps Center for Health Services Research at UNC Chapel Hill, rapidly created and conducted a needs assessment of PCPs in western North Carolina (WNC). Methods: A group of twenty volunteers conducted a telephone survey of PCPs in a 16 county region of WNC. Practices were asked about their COVID-19 testing and telehealth offerings, PPE adequacy, and capacity to continue serving patients. The survey's emergency alert feature linked practices to immediate support. Descriptive data were generated to identify regional needs. Results: Out of 110 practices, 48 (43.6%) offered COVID-19 testing, with testing more common in rural counties (56.3% vs 33.9%). Telehealth services, including phone-only visits, were offered by almost all practices (91.8%). PPE needs included N-95 respirators (49.1%), face shields (45.5%), and staff gowns (38.2%). Rural practices were more likely to report the need for PPE. Assistance was requested for staff member childcare (34.5%) and providing or billing for telehealth (31.8%). The most urgent practice requests were related to finances, PPE, and telehealth. MAHEC's Practice Support team linked practices to virtual coaching, tip sheets, case-based video didactics and communication forums, and newsletters. Conclusion: During a pandemic, it is crucial to ensure that PCPs can continue to serve their patients. A rapid needs assessment of PCPs can allow for immediate and ongoing support that matches regional and practice-specific needs. Rural practices may require more assistance than their urban counterparts. Our rapid survey process jumpstarted a statewide system for enhanced communications with PCPs to better prepare for future emergencies.

Usability Testing of an Electronic Patient-Reported Outcome System for Survivors of Critical Illness.

BACKGROUND: Web-based electronic patient-reported outcomes (ePRO) measures are increasingly used to facilitate patient-centered health assessments. However, it is unknown if ePRO completion is feasible for recently ill intensive care unit (ICU) survivors and their families. OBJECTIVE: To develop and evaluate the usability of a novel ePRO system (ePRO to Support People and Enhance Recovery [ePROSPER]) among ICU survivors and their families within an ongoing clinical trial. METHODS: Paper-based PROs were iteratively adapted to electronic forms (ePROs). Then, the usability of ePROSPER was assessed among 60 patients, their family members, and PRO and programming experts via questionnaires (eg, Systems Usability Scale), "think aloud" open-ended feedback, task completion times, and error rates. RESULTS: Input from patients and their families was used to incorporate user-experience modifications into ePROSPER. This feedback also led to inclusion of automated reminders for questionnaire completion and real-time alerts for staff triggered by high symptom levels. Median usability scores increased over testing cycles from 40 to 73 to 95, nearing the maximum score and showing excellent usability. All users completed ePROSPER within 20 minutes; 87% preferred it to a written version. ePROSPER was then implemented in a clinical trial without data errors. CONCLUSIONS: Automated ePRO systems can be successfully integrated in a post-ICU clinical trial setting. The value of integrating such systems in direct clinical care should be assessed in future studies.

Development and usability testing of a Web-based decision aid for families of patients receiving prolonged mechanical ventilation.

BACKGROUND: Web-based decision aids are increasingly important in medical research and clinical care. However, few have been studied in an intensive care unit setting. The objectives of this study were to develop a Web-based decision aid for family members of patients receiving prolonged mechanical ventilation and to evaluate its usability and acceptability. METHODS: Using an iterative process involving 48 critical illness survivors, family surrogate decision makers, and intensivists, we developed a Web-based decision aid addressing goals of care preferences for surrogate decision makers of patients with prolonged mechanical ventilation that could be either administered by study staff or completed independently by family members (Development Phase). After piloting the decision aid among 13 surrogate decision makers and seven intensivists, we assessed the decision aid's usability in the Evaluation Phase among a cohort of 30 surrogate decision makers using the Systems Usability Scale (SUS). Acceptability was assessed using measures of satisfaction and preference for electronic Collaborative Decision Support (eCODES) versus the original printed decision aid. RESULTS: The final decision aid, termed 'electronic Collaborative Decision Support', provides a framework for shared decision making, elicits relevant values and preferences, incorporates clinical data to personalize prognostic estimates generated from the ProVent prediction model, generates a printable document summarizing the user's interaction with the decision aid, and can digitally archive each user session. Usability was excellent (mean SUS, 80 ± 10) overall, but lower among those 56 years and older (73 ± 7) versus those who were younger (84 ± 9); p = 0.03. A total of 93% of users reported a preference for electronic versus printed versions. CONCLUSIONS: The Web-based decision aid for ICU surrogate decision makers can facilitate highly individualized information sharing with excellent usability and acceptability. Decision aids that employ an electronic format such as eCODES represent a strategy that could enhance patient-clinician collaboration and decision making quality in intensive care.

Engineering and therapeutic application of single-chain bivalent TGF-ß family traps.

Deregulation of TGF-ß superfamily signaling is a causative factor in many diseases. Here we describe a protein engineering strategy for the generation of single-chain bivalent receptor traps for TGF-ß superfamily ligands. Traps were assembled using the intrinsically disordered regions flanking the structured binding domain of each receptor as "native linkers" between two binding domains. This yields traps that are approximately threefold smaller than antibodies and consists entirely of native receptor sequences. Two TGF-ß type II receptor-based, single-chain traps were designed, termed (TßRII)2 and (TßRIIb)2, that have native linker lengths of 35 and 60 amino acids, respectively. Both single-chain traps exhibit a 100 to 1,000 fold higher in vitro ligand binding and neutralization activity compared with the monovalent ectodomain (TßRII-ED), and a similar or slightly better potency than pan-TGF-ß-neutralizing antibody 1D11 or an Fc-fused receptor trap (TßRII-Fc). Despite its short in vivo half-life (<1 hour), which is primarily due to kidney clearance, daily injections of the (TßRII)2 trap reduced the growth of 4T1 tumors in BALB/c mice by 50%, an efficacy that is comparable with 1D11 (dosed thrice weekly). In addition, (TßRII)2 treatment of mice with established 4T1 tumors (100 mm(3)) significantly inhibited further tumor growth, whereas the 1D11 antibody did not. Overall, our results indicate that our rationally designed bivalent, single-chain traps have promising therapeutic potential.

Transient expression and purification of chimeric heavy chain antibodies.

Monoclonal antibodies have been successfully engineered as approved therapeutics. However, their large size is considered a major factor preventing them from having a more efficient tissue penetration. As the first step to establish a possibly more efficient antibody platform, we present here transient expression, purification and characterization of six chimeric heavy chain antibodies (cHCAbs), or fusion of camelid single domain antibodies (sdAbs) to human fragment crystallizable (Fc). All six HCAbs have a MW of approximately 80 kDa, expressed well in a HEK293 expression system and have G0, G1 and G2 types of glycosylation. The transient expression also provided a very fast way to generate high milligram to low gram amount of proteins for in vitro assays and preliminary animal studies.

Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells.

BACKGROUND: Mammalian cells are becoming the prevailing expression system for the production of recombinant proteins because of their capacity for proper protein folding, assembly, and post-translational modifications. These systems currently allow high volumetric production of monoclonal recombinant antibodies in the range of grams per litre. However their use for large-scale expression of cytokines typically results in much lower volumetric productivity. RESULTS: We have engineered a HEK293 cell clone for high level production of human recombinant glycosylated IFNalpha2b and developed a rapid and efficient method for its purification. This clone steadily produces more than 200 mg (up to 333 mg) of human recombinant IFNalpha2b per liter of serum-free culture, which can be purified by a single-step cation-exchange chromatography following media acidification and clarification. This rapid procedure yields 98% pure IFNalpha2b with a recovery greater than 70%. Purified IFNalpha2b migrates on SDS-PAGE as two species, a major 21 kDa band and a minor 19 kDa band. N-terminal sequences of both forms are identical and correspond to the expected mature protein. Purified IFNalpha2b elutes at neutral pH as a single peak with an apparent molecular weight of 44,000 Da as determined by size-exclusion chromatography. The presence of intramolecular and absence of intermolecular disulfide bridges is evidenced by the fact that non-reduced IFNalpha2b has a greater electrophoretic mobility than the reduced form. Treatment of purified IFNalpha2b with neuraminidase followed by O-glycosidase both increases electrophoretic mobility, indicating the presence of sialylated O-linked glycan. A detailed analysis of glycosylation by mass spectroscopy identifies disialylated and monosialylated forms as the major constituents of purified IFNalpha2b. Electron transfer dissociation (ETD) shows that the glycans are linked to the expected threonine at position 106. Other minor glycosylated forms and non-sialylated species are also detected, similar to IFNalpha2b produced naturally by lymphocytes. Further, the HEK293-produced IFNalpha2b is biologically active as shown with reporter gene and antiviral assays. CONCLUSION: These results show that the HEK293 cell line is an efficient and valuable host for the production of biologically active and glycosylated human IFNalpha2b.

Scalable serum-free production of recombinant adeno-associated virus type 2 by transfection of 293 suspension cells.

Recombinant adeno-associated virus (rAAV) has emerged in recent years as a promising gene therapy vector that may be used in the treatment of diverse human diseases. The major obstacle to broadening the usage of rAAV vectors remains the limited capacity of available production systems to provide sufficient rAAV quantities for preclinical and clinical trials. The impracticality of expanding commonly used adherent cell lines represents a limitation to large-scale production. This paper describes successful productions of rAAV type 2 using suspension-growing human embryonic kidney (HEK293) cells in serum-free medium. The developed process, based on triple transfection employing polyethylenimine (PEI) as DNA transporter, allowed for a serum-free production of AAV, yielding viral vector titer up to 4.5x10(11) infectious viral particles (IVP) in a 3.5-L bioreactor. A maximum ratio of VG:IVP in the order of 200:1 was obtained, indicating the efficient encapsidation of viral vectors in HEK293 cells. The effect of varying the ratio of three plasmids and the influence of cell density at transfection were studied. The conditioned medium did not limit or inhibit the rAAV production; therefore, the elimination of the medium exchange step before or after transfection greatly simplified the scale-up of rAAV production. The cell-specific viral titers obtained in bioreactor suspension cultures were similar or higher than those obtained with control adherent cell cultures which further supported the scalability of the process. From multiple aspects including process simplicity, scalability, and low operating costs, this transfection method appears to be the most promising technology for large-scale production of rAAV.

Purification and characterization of a recombinant G-protein-coupled receptor, Saccharomyces cerevisiae Ste2p, transiently expressed in HEK293 EBNA1 cells.

The production of milligram quantities of purified, active, folded membrane protein from heterologous expression systems remains a general challenge due to intrinsically low expression levels, misfolding, and instability. Here we report the overexpression and purification of milligram quantities of functional Saccharomyces cerevisiae G-protein-coupled receptor, Ste2p, from transiently transfected human embryonic kidney 293 EBNA1 cells. Fluorescent microscopy indicates localization of Ste2p-GFP and Fc-Ste2p-GFP fusion receptors to the cell membrane. Up to 2 mg (approximately 10 pmol/million cells) of the Fc-Ste2p-GFP fusion and 1 mg of a Ste2p-Strep-TagII/(His)8-tagged version were purified per liter of culture following protein A-Sepharose and Talon metal affinity chromatography, respectively. Two distinct fluorescent labels, the hydrophobic 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) and the more hydrophilic fluorescein-5-maleimide (FM), were individually attached to the C-terminus of the alpha-mating factor ligand by addition of a reactive cysteine residue to produce active fluorescent pheromones. In vitro fluorescent ligand binding assays demonstrated that a high percentage of the recombinant purified receptor is correctly folded and able to bind ligand. KD values of 34 +/- 3 and 300 +/- 20 nM were observed respectively for the CPM- and FM-labeled ligands. These results combined with blue-shifted emission peaks and loss of fluorescent quenching observed for both fluorescent-labeled Cys alpha-factors when bound to receptor support a model in which the C-terminus of the ligand is packed in a hydrophobic pocket at the interface between the transmembrane and extracellular loop domains. Overall, we present an efficient system for recombinant production of milligram quantities of purified Ste2p in a biologically active form with applications to future structure and functional studies.

Purification of recombinant proteins from mammalian cell culture using a generic double-affinity chromatography scheme.

Transient transfection of mammalian cells has proven to be a useful technique for the rapid production of recombinant proteins because of its ability to produce milligram quantities within 2 weeks following cloning of their corresponding cDNA. This rapid production also requires a fast and efficient purification scheme that can be applied generically, typically through the use of affinity tags such as the polyhistidine-tag for capture by immobilized metal-affinity chromatography (IMAC) or the Strep-tag II, which binds to the StrepTactin affinity ligand. However, one-step purification using either of these tags has disadvantages in terms of yield, elution conditions, and purity. Here, we show that the addition of both Strep-tag-II and (His)(8) to the C-terminal of r-proteins allows efficient purification by consecutive IMAC and StrepTactin affinity. This approach has been successfully demonstrated using the intracellular protein DsRed, as well as two secreted proteins, secreted alkaline phosphatase (SEAP) and vascular endothelial growth factor (VEGF), all produced by transient transfection of HEK293-EBNA1 cells in medium supplemented with bovine calf serum. All proteins were purified to >99% homogeneity with yields varying from 29 to 81%.

Combined inhibition of PDGF and VEGF receptors by ellagic acid, a dietary-derived phenolic compound.

The vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptors play essential and complementary roles in angiogenesis and combined inhibition of these receptors has been shown to result in potent antitumor activity in vivo. In this study, we report that ellagic acid (EA), a natural polyphenol found in fruits and nuts, inhibits VEGF-induced phosphorylation of VEGFR-2 in endothelial cell (EC) as well as PDGF-induced phosphorylation of PDGFR in smooth muscle cells, leading to the inhibition of downstream signaling triggered by these receptors. EA also specifically inhibited VEGF-induced migration of ECs as well as their differentiation into capillary-like tubular structures and abolished PDGF-dependent smooth muscle cell migration. Interestingly, EA presents a greater selectivity for normal cells than for tumor cells since the migration of the U87 and HT1080 cell lines were much less affected by this molecule. The identification of EA as a naturally occurring dual inhibitor of VEGF and PDGF receptors suggests that this molecule possesses important antiangiogenic properties that may be helpful for the prevention and treatment of cancer.

Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency.

Large-scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins (r-proteins). As many r-proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted, a cost-effective serum-free culture medium that allows their efficient expression and purification is required. In an attempt to design such a serum-free medium, the effect of nine protein hydrolysates on cell proliferation, transfection efficiency, and volumetric productivity was evaluated using green fluorescent protein (GFP) and human placental secreted alkaline phosphate (SEAP) as reporter genes. The suspension growing, serum-free adapted HEK293SF-3F6 cell line was stably transfected with an EBNA1-expression vector to increase protein expression when using EBV oriP bearing plasmids. Compared to our standard serum-free medium, concomitant addition of the gelatin peptone N3 and removal of BSA slightly enhanced transfection efficiency and significantly increased volumetric productivity fourfold. Using the optimized medium formulation, transfection efficiencies between 40-60% were routinely obtained and SEAP production reached 18 mg/L(-1). To date, we have successfully produced and purified over fifteen r-proteins from 1-14-L bioreactors using this serum-free system. As examples, we describe the scale-up of two secreted his-tagged r-proteins Tie-2 and Neuropilin-1 extracellular domains (ED) in bioreactors. Each protein was successfully purified to >95% purity following a single immobilized metal affinity chromatography (IMAC) step. In contrast, purification of Tie-2 and Neuropilin-1 produced in serum-containing medium was much less efficient. Thus, the use of our new serum-free EBNA1 cell line with peptone-enriched serum-free medium significantly improves protein expression compared to peptone-less medium, and significantly increases their purification efficiency compared to serum-containing medium. This eliminates labor-intensive and expensive chromatographic steps, and allows for the simple, reliable, and extremely fast production of milligram amounts of r-proteins within 5 days posttransfection.