Early plasma interferon-ß levels as a predictive marker of COVID-19 severe clinical events in adult patients.

We assessed relationships between early peripheral blood type I interferons (IFN) levels, clinical new early warning scores (NEWS), and clinical outcomes in hospitalized coronavirus disease-19 (COVID-19) adult patients. Early IFN-ß levels were lower among patients who further required intensive care unit (ICU) admission than those measured in patients who did not require an ICU admission during severe acute respiratory syndrome coronavirus type 2 infection. IFN-ß levels were inversely correlated with NEWS only in the subgroup of patients who further required ICU admission. To assess whether peripheral blood IFN-ß levels could be a potential relevant biomarker to predict further need for ICU admission, we performed receiver operating characteristic (ROC) curve analyses that showed for all study patients an area under ROC curve of 0.77 growing to 0.86 (p = 0.003) when the analysis was restricted to a subset of patients with NEWS ≥5 at the time of hospital admission. Overall, our findings indicated that early peripheral blood IFN-ß levels might be a relevant predictive marker of further need for an ICU admission in hospitalized COVID-19 adult patients, specifically when clinical score (NEWS) was graded as upper than 5 at the time of hospital admission.

Optimizing the Semisynthesis towards glycosylated interferon-ß-polypeptide by utilizing bacterial protein expression and chemical modification.

The synthesis of a sufficient amount of homogeneous glycoprotein is of great interest because natural glycoproteins show considerable heterogeneity in oligosaccharide structures, making the studies on glycan structure-function relationship difficult. Herein, we report optimized methods that can accelerate the semisynthesis of homogeneous glycoproteins based on recombinant expression and chemical conversion. Peptide thioesters and peptides with Cys residues at their N-terminals are necessary intermediates to perform native chemical ligation. We successfully performed thioesterification for a peptide prepared in E. coli via Cys-cyanylation at its C-terminal followed by hydrazinolysis and acidic thiolysis. These optimized conditions could tolerate an acid labile Thz protected Cys at the N-terminal of a peptide-hydrazide and specific cyanylation of the C-terminal Cys to yield a peptide thioester. To reduce the amount of precious oligosaccharide that is required in the conventional SPPS method, an improved liquid phase glycopeptide coupling was also optimized in a good yield (46% over four steps). Lastly, chemoselective protection of the internal cysteines and activation of the N-terminal cysteine were optimized toward a long peptide prepared in E. coli. By using these strategies, a full-length interferon-ß glycosyl polypeptide as a model was successfully obtained.

Structural basis for IFN antagonism by human respiratory syncytial virus nonstructural protein 2.

Human respiratory syncytial virus (RSV) nonstructural protein 2 (NS2) inhibits host interferon (IFN) responses stimulated by RSV infection by targeting early steps in the IFN-signaling pathway. But the molecular mechanisms related to how NS2 regulates these processes remain incompletely understood. To address this gap, here we solved the X-ray crystal structure of NS2. This structure revealed a unique fold that is distinct from other known viral IFN antagonists, including RSV NS1. We also show that NS2 directly interacts with an inactive conformation of the RIG-I-like receptors (RLRs) RIG-I and MDA5. NS2 binding prevents RLR ubiquitination, a process critical for prolonged activation of downstream signaling. Structural analysis, including by hydrogen-deuterium exchange coupled to mass spectrometry, revealed that the N terminus of NS2 is essential for binding to the RIG-I caspase activation and recruitment domains. N-terminal mutations significantly diminish RIG-I interactions and result in increased IFNß messenger RNA levels. Collectively, our studies uncover a previously unappreciated regulatory mechanism by which NS2 further modulates host responses and define an approach for targeting host responses.

Stability and Activity of the Hyperglycosylated Human Interferon-ß R27T Variant.

A hyperglycosylated recombinant human interferon-ß (rhIFN-ß) R27T mutant was established to improve relapsing-remitting multiple sclerosis (RRMS) in our previous study. We focused on the stability of the R27T mutant throughout its production lifetime, including culture, purification, and storage before formulation prior to clinical use. Herein, we address the stability of this protein during optimized culture and purification processes. Additionally, we employed artificial stress conditions during culture and purification to characterize R27T instability. Although, among total R27T, relative native R27T ratio displayed transiently low even under optimized production process, the ratio was recovered by the end of the overall production process, suggesting that culture and purification processes are optimized. Artificial stress during culture and purification processes resulted in degradation of R27T acidic and basic variants, and mismatched disulfide bonds in no-aggregated forms as well as in the aggregated form. The presence of disulfide bond exchange without aggregation in the unfolded/misfolded state could be a novel finding for rhIFN-ß products. The results provide meaningful information for the comprehensive evaluation of the stability of the R27T variant.

Highly specific interaction of monomeric S100P protein with interferon beta.

S100 proteins are EF-hand calcium-binding proteins of vertebrates exerting numerous intra- and extracellular actions and involved into multiple diseases. Some of S100 proteins serve as extracellular damage signals via interaction with receptors. Although several S100 proteins directly bind specific cytokines, this phenomenon remains underexplored. Using chemical crosslinking, intrinsic fluorescence and surface plasmon resonance spectroscopies, we show that S100P protein interacts with interferon beta (IFN-ß) depending on calcium level and oligomeric state of S100P. Dimeric Ca2+-loaded S100P binds IFN-ß with equilibrium dissociation constants, Kd, of 0.05-0.6 µM. S100P monomerization favors this interaction decreasing Kd values down to 0.3-2 nM. Calcium depletion drastically lowers S100P affinity to IFN-ß. Other related EF-hand proteins studied (calmodulin, α-parvalbumin and S100G) do not bind IFN-ß, thereby confirming selectivity of the S100P - IFN-ß interaction. Crystal violet assay reveals that the S100P binding suppresses IFN-ß cytotoxicity to MCF-7 breast cancer cells. Since several cancers (breast, colon, lung, liver, etc.) exhibit dysregulated functioning of S100P and IFN-ß, their interaction could be relevant to the cancer progression and directed therapeutic interventions.

Real-time 2-5A kinetics suggest that interferons ß and λ evade global arrest of translation by RNase L.

Cells of all mammals recognize double-stranded RNA (dsRNA) as a foreign material. In response, they release interferons (IFNs) and activate a ubiquitously expressed pseudokinase/endoribonuclease RNase L. RNase L executes regulated RNA decay and halts global translation. Here, we developed a biosensor for 2',5'-oligoadenylate (2-5A), the natural activator of RNase L. Using this biosensor, we found that 2-5A was acutely synthesized by cells in response to dsRNA sensing, which immediately triggered cellular RNA cleavage by RNase L and arrested host protein synthesis. However, translation-arrested cells still transcribed IFN-stimulated genes and secreted IFNs of types I and III (IFN-ß and IFN-λ). Our data suggest that IFNs escape from the action of RNase L on translation. We propose that the 2-5A/RNase L pathway serves to rapidly and accurately suppress basal protein synthesis, preserving privileged production of defense proteins of the innate immune system.

Monitoring of Glycoprotein Quality Control System with a Series of Chemically Synthesized Homogeneous Native and Misfolded Glycoproteins.

The glycoprotein quality control (GQC) system in the endoplasmic reticulum (ER) effectively uses chaperone-type enzymes and lectins such as UDP-glucose:glycoprotein glucosyltransferase (UGGT), calnexin (CNX), calreticulin (CRT), protein disulfide bond isomerases (ERp57 or PDIs), and glucosidases to generate native-folded glycoproteins from nascent glycopolypeptides. However, the individual processes of the GQC system at the molecular level are still unclear. We chemically synthesized a series of several homogeneous glycoproteins bearing M9-high-mannose type oligosaccharides (M9-glycan), such as erythropoietin (EPO), interferon-ß (IFN-ß), and interleukin 8 (IL8) and their misfolded counterparts, and used these glycoprotein probes to better understand the GQC process. The analyses by high performance liquid chromatography and mass spectrometer clearly showed refolding processes from synthetic misfolded glycoproteins to native form through folding intermediates, allowing for the relationship between the amount of glucosylation and the refolding of the glycoprotein to be estimated. The experiment using these probes demonstrated that GQC system isolated from rat liver acts in a catalytic cycle regulated by the fast crosstalk of glucosylation/deglucosylation in order to accelerate refolding of misfolded glycoproteins.

Biocompatible Coatings from Smart Biopolymer Nanoparticles for Enzymatically Induced Drug Release.

Nanoparticles can be used as a smart drug delivery system, when they release the drug only upon degradation by specific enzymes. A method to create such responsive materials is the formation of hydrogel nanoparticles, which have enzymatically degradable crosslinkers. Such hydrogel nanoparticles were prepared by ionotropic gelation sodium alginate with lysine-rich peptide sequences-either α-poly-L-lysine (PLL) or the aggrecanase-labile sequence KKKK-GRD-ARGSV↓NITEGE-DRG-KKKK. The nanoparticle suspensions obtained were analyzed by means of dynamic light scattering and nanoparticle tracking analysis. Degradation experiments carried out with the nanoparticles in suspension revealed enzyme-induced lability. Drugs present in the polymer solution during the ionotropic gelation can be encapsulated in the nanoparticles. Drug loading was investigated for interferon-ß (IFN-ß) as a model, using a bioluminescence assay with MX2Luc2 cells. The encapsulation efficiency for IFN-ß was found to be approximately 25%. The nanoparticles suspension can be used to spray-coat titanium alloys (Ti-6Al-4V) as a common implant material. The coatings were proven by ellipsometry, reflection-absorption infrared spectroscopy, and X-ray photoelectron spectroscopy. An enzyme-responsive decrease in layer thickness is observed due to the degradation of the coatings. The Alg/peptide coatings were cytocompatible for human gingival fibroblasts (HGFIB), which was investigated by CellTiterBlue and lactate dehydrogenase (LDH) assay. However, HGFIBs showed poor adhesion and proliferation on the Alg/peptide coatings, but these could be improved by modification of the alginate with a RGD-peptide sequence. The smart drug release system presented can be further tailored to have the right release kinetics and cell adhesion properties.

IFNb of black carp functions importantly in host innate immune response as an antiviral cytokine.

Type I interferons (IFN-Is) play an important role in the antiviral immune response in teleost fishes. In this study, one type I interferon (bcIFNb) from black carp (Mylopharyngodon piceus) has been cloned and characterized. The full-length cDNA of bcIFNb gene consists of 806 nucleotides and the predicted bcIFNb protein contains 188 amino acids. Basing on the cysteine number and evolutionary position, bcIFNb was classified into group II type I IFN. q-PCR analysis demonstrated that bcIFNb mRNA level varied in vivo and ex vivo in response to different stimuli. bcIFNb was detected in both the whole cell lysate and the supernatant media of HEK293T cells or EPC cells transfected with bcIFNb through immunoblot assay. IFN stimulated genes (ISGs) were greatly upregulated when the host cells were treated with the bcIFNb-containing conditioned media. EPC cells showed greatly enhanced antiviral ability when the cells were transfected with bcIFNb or treated with the bcIFNb-containing conditioned media before GCRV or SVCV infection. Glycosidase digestion analysis determined that bcIFNb was modified with N-linked glycosylation, which occurred on the Asn (N) of 92 site of this cytokine. The un-glycosylated mutant bcIFNb-N92Q presented the similar antiviral ability as that of wild type bcIFNb, which demonstrated that N-linked glycosylation did not contribute directly to the antiviral property of this fish cytokine.

Direct Antimicrobial Activity of IFN-ß.

Type I IFNs are a cytokine family essential for antiviral defense. More recently, type I IFNs were shown to be important during bacterial infections. In this article, we show that, in addition to known cytokine functions, IFN-ß is antimicrobial. Parts of the IFN-ß molecular surface (especially helix 4) are cationic and amphipathic, both classic characteristics of antimicrobial peptides, and we observed that IFN-ß can directly kill Staphylococcus aureus Further, a mutant S. aureus that is more sensitive to antimicrobial peptides was killed more efficiently by IFN-ß than was the wild-type S. aureus, and immunoblotting showed that IFN-ß interacts with the bacterial cell surface. To determine whether specific parts of IFN-ß are antimicrobial, we synthesized IFN-ß helix 4 and found that it is sufficient to permeate model prokaryotic membranes using synchrotron x-ray diffraction and that it is sufficient to kill S. aureus These results suggest that, in addition to its well-known signaling activity, IFN-ß may be directly antimicrobial and be part of a growing family of cytokines and chemokines, called kinocidins, that also have antimicrobial properties.

Novel pegylated interferon-ß as strong suppressor of the malignant ascites in a peritoneal metastasis model of human cancer.

Malignant ascites manifests as an end-stage event during the progression of a number of cancers and lacks a generally accepted standard therapy. Interferon-ß (IFN-ß) has been used to treat several cancer indications; however, little is known about the efficacy of IFN-ß on malignant ascites. In the present study, we report on the development of a novel, engineered form of human and murine IFN-ß, each conjugated with a polyethylene glycol molecule (PEG-hIFN-ß and PEG-mIFN-ß, respectively). We provide evidence that these IFN-ß molecules retain anti-viral potency comparable to unmodified IFN-ß in vitro and manifested improved pharmacokinetics in vivo. Interestingly, PEG-mIFN-ß significantly inhibited the accumulation of ascites fluid and vascular permeability of the peritoneal membrane in models of ovarian cancer and gastric cancer cell xenograft mice. We further show that PEG-hIFN-ß directly suppresses VEGF165 -induced hyperpermeability in a monolayer of human vascular endothelial cells and that PEG-mIFN-ß enhanced gene expression for a number of cell adhesion related molecules in mouse vascular endothelial cells. Taken together, these findings unveil a hitherto unrecognized potential of IFN-ß in maintaining vascular integrity, and provide proof-of-mechanism for a novel and long-acting pegylated hIFN-ß for the therapeutic treatment of malignant ascites.

Rational design of glycoengineered interferon-ß analogs with improved aggregation state: experimental validation.

Recombinant human interferon-ß (rhIFN-ß) used clinically has lower efficacy than expected due to protein instabilities such as aggregation. Increasing molecular stability, glycoengineering has been used to improve clinical efficacy for a number of therapeutics; however, often labor-intensive trail-and-error approaches are used to identify additional glycosylation sites. In this study two rhIFN-ß analogs with one additional glycosylation site, L6T and S75N, identified by a rational in silico approach, were characterized. These rhIFN-ß analogs were synthesized in parallel with a Chinese hamster ovary (CHO) codon-optimized natural human IFN-ß (Opt-IFN-ß) and expressed in CHO cells using the same expression system. The molecular weights for both analogs were observed to be higher than Opt-IFN-ß, consistent with hyper-glycosylation. The in vitro biological assay showed the hyper-glycosylated analogs and the Opt-IFN-ß had similar activity. The aggregation studies demonstrated that both analogs had lower tendencies to aggregate compared to the Opt-IFN-ß. These experimental studies validate the in silico strategy to predict suitable glycosylation sites that would be glycosylated, while maintaining biological function. Moreover, this work describes hyper-glycosylated rhIFN-ß analogs with improved solubility (i.e. lower aggregation). These findings, together with the rational in silico design, will allow us to increase protein glycosylation with the goal to enhance therapeutic efficacy.

Use of a non-covalent cell-penetrating peptide strategy to enhance the nasal delivery of interferon beta and its PEGylated form.

The conjugation of therapeutic proteins to polyethylene glycol (PEG) is known as PEGylation. It improves their retention in the body and reduces the frequency of injections. Development of noninvasive delivery systems for biopharmaceuticals can improve the patients' quality of life. The present study aimed to evaluate the cell-penetrating peptides (CPPs), which act as bioenhancers, for the nasal delivery of protein drug interferon beta (IFN-ß) and its PEGylated form (PEG-IFN-ß). The ability of CPPs to enhance the nasal mucosal absorption of unmodified IFN-ß was assessed in rats. It was shown that only d-amino acid forms of amphipathic CPPs, penetratin and PenetraMax significantly enhanced the nasal absorption of IFN-ß. Especially, D-penetratin (up to 2mM) enhanced the absorption of INF-ß in a dose-dependent manner. The maximum absolute bioavailability reached 8.26% following in situ nasal coadministration of IFN-ß with d-penetratin (2mM). Furthermore, it was found that the coadministration of d-penetratin also facilitated the nasal absorption of PEG-IFN-ß, which remained in the circulation for more than 6h. Moreover, the toxicity assessments showed no damage to the epithelial membranes after nasal administration of CPPs including penetratin and PenetraMax. Altogether, this study provides the first evidence that the noncovalent coadministration of PEGylated proteins with CPPs could be a potent strategy for the noninvasive and sustained nasal delivery of therapeutic proteins.

Evaluation of Incremental Siliconization Levels on Soluble Aggregates, Submicron and Subvisible Particles in a Prefilled Syringe Product.

The evaluation of stability with respect to particles in prefilled syringes is complicated by the presence of silicone oil. The mobility, colloidal characteristics, and kinetic instability of silicone oil in contact with a protein formulation may be influenced in unpredictable ways by pharmaceutical variables, storage, and handling conditions. To provide insight into the impact of these variables on silicone oil originating specifically from the siliconized prefillable syringe (PFS), a series of studies were conducted at incremental syringe barrel siliconization levels. Size-exclusion chromatography and particle counting methods were used to quantitate soluble aggregates and submicron and subvisible particles in peginterferon beta-1a in a PFS siliconized with a fixed nozzle spray-on siliconization process. The effect of silicone oil on the peginterferon beta-1a molecule was examined under pharmaceutically relevant conditions, accelerated degradation, and under denaturing conditions. Resonant mass measurement was used to discriminate silicone oil from protein particles establishing that silicone oil does not mask adverse trends in non-silicone oil particles. The peginterferon beta-1a molecule was shown to be stable in the presence of silicone oil and robust with respect to the formation of soluble aggregates and submicron and subvisible particles in its PFS siliconized over the range of 0-1.2 mg silicone oil per syringe barrel.

High Resolution CZE-MS Quantitative Characterization of Intact Biopharmaceutical Proteins: Proteoforms of Interferon-ß1.

New and improved methods are required for the enhanced characterization of complex biopharmaceuticals, especially those with charge and glycan heterogeneity. High resolution separation and mass spectrometry (MS) analysis of intact proteoforms can contribute significantly to the characterization of such proteins, many of which are glycoproteins. Here, we report on capillary zone electrophoresis (CZE) coupled via a commercial CESI sheathless interface to an Orbitrap ELITE MS for the intact analysis of recombinant human interferon-ß1 (Avonex, rhIFN-ß1), a biopharmaceutical with complex glycosylation at a single N-linked site. Using a cross-linked polyethylenimine coating, column efficiencies between 350,000 and 450,000 plates were produced, allowing separation based on charge and subtle hydrodynamic volume differences. A total of 138 proteoforms were found, and 55 were quantitated. Charge species due to deamidation and sialylation were separated by CZE. Given the high column efficiency, isobaric positional isomers of a single sialic acid on biantennary glycan antennae were resolved. Further, triantennary isomers (antenna on α(1-3) or α(1-6) arms) were separated and confirmed by exoglycosidase digestion. Proteoforms of the N-terminal cleavage of methionine were detected by precursor molecular weight and top-down ETD and HCD analysis of the reduced protein. Quantitative analysis suggested potential correlations between the methionine loss with the relative amount of the deamidation, as well as the level of deamidation with glycan structure. We demonstrate that high resolution CZE separation of intact glycoprotein species coupled to MS has significant potential for the in-depth characterization and quantitative analysis of biopharmaceutical proteoforms.

Prediction of Spontaneous Protein Deamidation from Sequence-Derived Secondary Structure and Intrinsic Disorder.

Asparagine residues in proteins undergo spontaneous deamidation, a post-translational modification that may act as a molecular clock for the regulation of protein function and turnover. Asparagine deamidation is modulated by protein local sequence, secondary structure and hydrogen bonding. We present NGOME, an algorithm able to predict non-enzymatic deamidation of internal asparagine residues in proteins in the absence of structural data, using sequence-based predictions of secondary structure and intrinsic disorder. Compared to previous algorithms, NGOME does not require three-dimensional structures yet yields better predictions than available sequence-only methods. Four case studies of specific proteins show how NGOME may help the user identify deamidation-prone asparagine residues, often related to protein gain of function, protein degradation or protein misfolding in pathological processes. A fifth case study applies NGOME at a proteomic scale and unveils a correlation between asparagine deamidation and protein degradation in yeast. NGOME is freely available as a webserver at the National EMBnet node Argentina, URL: http://www.embnet.qb.fcen.uba.ar/ in the subpage "Protein and nucleic acid structure and sequence analysis".

Characterization of intact protein conjugates and biopharmaceuticals using ion-exchange chromatography with online detection by native electrospray ionization mass spectrometry and top-down tandem mass spectrometry.

Characterization of biopharmaceutical products is a challenging task, which needs to be carried out at several different levels (including both primary structure and conformation). An additional difficulty frequently arises due to the structural heterogeneity inherent to many protein-based therapeutics (e.g., extensive glycosylation or "designer" modifications such as chemical conjugation) or introduced postproduction as a result of stress (e.g., oxidation and deamidation). A combination of ion-exchange chromatography (IXC) with online detection by native electrospray ionization mass spectrometry (ESI MS) allows characterization of complex and heterogeneous therapeutic proteins and protein conjugates to be accomplished at a variety of levels without compromising their conformational integrity. The IXC/ESI MS measurements allow protein conjugates to be profiled by analyzing conjugation stoichiometry and the presence of multiple positional isomers, as well as to establish the effect of chemical modifications on the conformational integrity of each species. While mass profiling alone is not sufficient for identification of nonenzymatic post-translational modifications (PTMs) that result in a very small mass change of the eluting species (e.g., deamidation), this task can be completed using online top-down structural analysis, as demonstrated using stressed interferon-ß as an example. The wealth of information that can be provided by IXC/native ESI MS and tandem mass spectrometry (MS/MS) on protein-based therapeutics will undoubtedly make it a very valuable addition to the experimental toolbox of biopharmaceutical analysis.

Basal buffer systems for a newly glycosylated recombinant human interferon-ß with biophysical stability and DoE approaches.

The purpose of this study was to develop a basal buffer system for a biobetter version of recombinant human interferon-ß 1a (rhIFN-ß 1a), termed R27T, to optimize its biophysical stability. The protein was pre-screened in solution as a function of pH (2-11) using differential scanning calorimetry (DSC) and dynamic light scattering (DLS). According to the result, its experimental pI and optimal pH range were 5.8 and 3.6-4.4, respectively. Design of experiment (DoE) approach was developed as a practical tool to aid formulation studies as a function of pH (2.9-5.7), buffer (phosphate, acetate, citrate, and histidine), and buffer concentration (20 mM and 50 mM). This method employed a weight-based procedure to interpret complex data sets and to investigate critical key factors representing protein stability. The factors used were Tm, enthalpy, and relative helix contents which were obtained by DSC and Fourier Transform Infrared spectroscopy (FT-IR). Although the weights changed by three responses, objective functions from a set of experimental designs based on four buffers were highest in 20 mM acetate buffer at pH 3.6 among all 19 scenarios tested. Size exclusion chromatography (SEC) was adopted to investigate accelerated storage stability in order to optimize the pH value with susceptible stability since the low pH was not patient-compliant. Interestingly, relative helix contents and storage stability (monomer remaining) increased with pH and was the highest at pH 4.0. On the other hand, relative helix contents and thermodynamic stability decreased at pH 4.2 and 4.4, suggesting protein aggregation issues. Therefore, the optimized basal buffer system for the novel biobetter was proposed to be 20 mM acetate buffer at pH 3.8±0.2.

Stucturally Diverse Sesquiterpenes Produced by a Chinese Tibet Fungus Stereum hirsutum and Their Cytotoxic and Immunosuppressant Activities.

Two new heterodimeric sesquiterpenes, sterhirsutins C (1) and D (2), along with eight new sesquiterpenoid derivatives, sterhirsutins E--L (3-10), were isolated from the culture of Stereum hirsutum. The absolute configuration of 1 was assigned by a single-crystal X-ray diffraction experiment. Compounds 1 and 2 possessed an unprecedented chemical skeleton with a 5/5/5/6/9/4 fused ring system. Compound 10 is the first sesquiterpene coupled with a xanthine moiety. Compounds 1-10 showed cytotoxicity against K562 and HCT116 cell lines. Compound 9 induced autophagy in HeLa cells. Compound 5 inhibited the activation of IFNß promoter in Sendai virus infected cells.

Rational design of hyper-glycosylated interferon beta analogs: a computational strategy for glycoengineering.

Glycoengineering has been successfully used to improve the physicochemical and pharmaceutical properties of therapeutics. One aspect of glycoengineering is to introduce new N-linked glycosylation consensus sequences (Asn, X, Thr/Ser) into desirable positions in the peptide backbone by mutational insertion to generate proteins with increased sialic acid content. In the current work, human interferon beta (huIFN-ß) was used as a model to identify the potential positions for the addition of new N-glycosylation sites. A computational strategy was employed to predict the structural distortions and functional alterations that might be caused by the change in amino acid sequence. Accordingly, three-dimensional (3D) structures of the designed huIFN-ß analogs were generated by comparative modeling. Molecular dynamics (MD) simulation was carried out to assess the molecular stability and flexibility profile of the structures. Subsequently, for the purpose of glycoengineering huIFN-ß, analogs with 3D structures more similar to the wild-type huIFN-ß and exposed Asn residue in the new N-glycosylation site were identified. These modeling procedures indicated that the addition of the new N-glycosylation site in the loop regions had lower constraining effects on the tertiary structure of the protein. This computational strategy can be applied to avoid alterations in the 3D structure of proteins caused by changes in the amino acid sequences, when designing novel hyper-glycosylated therapeutics. This in turn reduces labor-intensive experimental analyses of each analog.