Physicochemical characterization of Remsima®

Remsima® (infliximab) was recently approved as the world's first biosimilar monoclonal antibody (mAb) in both the European Union and Korea. To achieve this, extensive physicochemical characterization of Remsima® in relation to Remicade® was conducted in order to demonstrate the highly similar properties between the two molecules. A multitude of state-of-the-art analyses revealed that Remsima® has identical primary as well as indistinguishable higher order structures compared with the original product. Monomer and aggregate contents of Remsima® were also found to be comparable with those of Remicade®. In terms of charge isoforms, although Remsima® was observed to contain slightly less basic variants than the original antibody, the difference was shown to be largely due to the presence of C-terminal lysine. On the other hand, this lysine was found to be rapidly clipped inside serum in vitro and in vivo, suggesting it has no effect on the biological potency or safety of the drug. Analysis of the glycan contents of the antibodies showed comparable glycan types and distributions. Recent results of clinical studies have further confirmed that the two antibody products are highly similar to each other. Based on this research as well as previous clinical and non-clinical comparability studies, Remsima® can be considered as a highly similar molecule to Remicade® in terms of physicochemical properties, efficacy, and safety for its final approval as a biosimilar product to Remicade®.     

Synthesis and characterization of pHLIP® coated gold nanoparticles

Novel approaches in synthesis of spherical and multispiked gold nanoparticles coated with polyethylene glycol (PEG) and pH Low Insertion Peptide (pHLIP^®) were introduced. The presence of a tumor-targeting pHLIP^® peptide in the nanoparticle coating enhances the stability of particles in solution and promotes a pH-dependent cellular uptake. The spherical particles were prepared with sodium citrate as a gold reducing agent to form particles of 7.0±2.5 nm in mean metallic core diameter and ～43 nm in mean hydrodynamic diameter. The particles that were injected into tumors in mice (21 µg of gold) were homogeneously distributed within a tumor mass with no staining of the muscle tissue adjacent to the tumor. Up to 30% of the injected gold dose remained within the tumor one hour post-injection. The multispiked gold nanoparticles with a mean metallic core diameter of 146.0±50.4 nm and a mean hydrodynamic size of ~161 nm were prepared using ascorbic acid as a reducing agent and disk-like bicelles as a template. Only the presence of a soft template, like bicelles, ensured the appearance of spiked nanoparticles with resonance in the near infrared region. The irradiation of spiked gold nanoparticles by an 805 nm laser led to the time- and concentration-dependent increase of temperature. Both pHLIP^® and PEG coated gold spherical and multispiked nanoparticles might find application in radiation and thermal therapies of tumors.     

Design and development of a new ambr250® bioreactor vessel for improved cell and gene therapy applications

The emergence of cell and gene therapies has generated significant interest in their clinical and commercial potential. However, these therapies are prohibitively expensive to manufacture and can require extensive time for development due to our limited process knowledge and understanding. The automated ambr250® stirred-tank bioreactor platform provides an effective platform for high-throughput process development. However, the original dual pitched-blade 20 mm impeller and baffles proved sub-optimal for cell therapy candidates that require suspension of microcarriers (e.g. for the culture of adherent human mesenchymal stem cells) or other particles such as activating Dynabeads® (e.g. for the culture of human T-cells). We demonstrate the development of a new ambr250® stirred-tank bioreactor vessel which has been designed specifically to improve the suspension of microcarriers/beads and thereby improve the culture of such cellular systems. The new design is unbaffled and has a single, larger elephant ear impeller. We undertook a range of engineering and physical characterizations to determine which vessel and impeller configuration would be most suitable for suspension based on the minimum agitation speed (N_JS) and associated specific power input (P/V)_JS. A vessel (diameter, T, = 60 mm) without baffles and incorporating a single elephant ear impeller (diameter 30 mm and 45° pitch-blade angle) was selected as it had the lowest (P/V)_JS and therefore potentially, based on Kolmogorov concepts, was the most flexible system. These experimentally-based conclusions were further validated firstly with computational fluid dynamic (CFD) simulations and secondly experimental studies involving the culture of both T-cells with Dynabeads® and hMSCs on microcarriers. The new ambr250® stirred-tank bioreactor successfully supported the culture of both cell types, with the T-cell culture demonstrating significant improvements compared to the original ambr250® and the hMSC-microcarrier culture gave significantly higher yields compared with spinner flask cultures. The new ambr250® bioreactor vessel design is an effective process development tool for cell and gene therapy candidates and potentially for autologous manufacture too.

     

Physiologically clotted fibrin – Preparation and characterization for tissue engineering and drug delivery applications

Fibrin used for biomedical applications is prepared by mixing concentrated solutions of fibrinogen and thrombin in presence of cross-linking agents such as Factor XIII or glutaraldehyde. The main drawbacks associated with this procedure include cost, complexity and time required for fibrin preparation. Hence, present study deals with the characterization of physiologically clotted fibrin (PF) for bone tissue engineering and drug delivery applications. For this the physico-chemical properties of PF were compared with those of the conventionally prepared fibrin (CF). Further MTT and haemolytic assays were performed for both PF and CF to compare their biocompatibility. The amount of alkaline phosphatase produced and calcium secreted by MG-63 cells in the presence of PF and CF were used to relate the osteogenic potency of PF with that of CF. Gallic acid, an anti-cancer drug was loaded within PF and CF and their role in drug delivery was compared.     

Establishment and characterization of lung co-culture spheroids for paclitaxel loaded Eudragit® RL 100 nanoparticle evaluation

3D cell cultures are regarded as a better and more relevant approach for screening drugs and therapeutics, particularly due to their likeness with the in vivo conditions. Spheroids offer an intermediate platform between in vitro and in vivo models, for conducting tumor-based investigations. In this study, a simple setup was developed for consistent generation of lung co-culture spheroids, which were developed using the cancer cell lines A549, NCI H460, and fibroblast cells WI-38. The potential of these spheroids for evaluating the toxicity of Eudragit® RL 100 nanoparticles (ENP) was explored. Monodisperse ENP, having the size range of 140–200 nm was prepared using the nanoprecipitation method. These were loaded with the poorly water-soluble anticancer drug paclitaxel. The evaluation of toxicity and uptake of drug-loaded ENP revealed that 2D monolayers were more sensitive to treatment than 3D spheroids. Within spheroids, co-cultures were more resistant to the treatment than monocultures. Overall, our findings demonstrated that the lung co-culture spheroids were a suitable model for accelerating the efficacy and toxicity-related investigations of novel drug delivery systems.     

Bioactivity and viscoelastic characterization of chitosan/bioglass® composite membranes

Membranes of chitosan (CTS) and composite membranes of CTS with bioglass are prepared by solvent casting. The composite membranes are shown to induce the precipitation of apatite upon immersion in SBF. The biomineralization process is followed by measuring the variation of the viscoelastic properties of the membranes immersed in SBF, both online and offline. Non-conventional DMA is used to measure the change in the storage modulus, E', and the loss factor, tan δ, as a function of the immersion in SBF. A simple model is used to estimate the E' of the apatite layer formed in vitro that is about 130?MPa. This work shows that innovate mechanical tests can be useful to characterize the mechanical performance of composites under physiological conditions.     

Modification of Esperase® by covalent bonding to Eudragit® polymers L 100 and S 100 for wool fibre surface treatment

The protease Esperase® was modified by covalent bonding with two grades of a reversible soluble-insoluble co-polymer of methacrylic acid and methyl-methacrylate, namely Eudragit® L 100 and Eudragit® S 100. The optimum reaction conditions and washing protocol were investigated and it was found that Esperase® modified with Eudragit® L 100 showed greater activity than if modified with Eudragit® S 100. This should be expected as there is a greater quantity of active sites, namely carboxyl groups, per mass of Eudragit® L 100 in comparison with Eudragit® S 100 to interact with the enzyme. Gel filtration confirmed that Eudragit® L 100 covalently bonded to Esperase®. Treatment of the modified Esperase® on wool showed that the enzyme modified with Eudragit® L 100 had greater activity towards the wool and appeared more effective in shrink resistant finishing.     

Phyto-assisted synthesis,characterization and applications of gold nanoparticles – A review

Nanotechnology is the formation, running and use of operation at the nanomaterial size scale (1–100 nm). Nanoscale materials can also be obtained by biological synthesis materials via eco-friendly green chemistry based technique. Current development and numerous strategies involved in the green synthesis of nanoparticles were focussed. This review mainly focused on plants which include scientific name, family name, common name, plant parts, its characterization, size and shape of the nanoparticles. Plant extract which was done experimentally gives its various characterization which leads to the identification of compounds of different nano size and shape. Biosynthesis of gold nanoparticles is in different shapes like spherical, rod, cubic, triangle and also in different sizes. Various application and importance of gold nanoparticles in numerous fields were discussed. The mark of the review is to provide an overview of recent learning in biosynthesized nanoparticles, its characterization and their potential applications.     

Synthesis, characterization and applications of graft copolymer (κ-carrageenan-g-vinylsulfonic acid)

The synthesis of graft copolymer (κ-carrageenan-g-vinylsulfonic acid) is carried out in nitrogen atmosphere using potassium peroxymonosulfate (PMS) and malonic acid (MA) as redox system. The effect of reaction variables including the concentration of vinylsulfonic acid 1.3×10(-2) to 6.7×10(-2) mol dm(-3), PMS 4×10(-3) to 20×10(-3) mol dm(-3), MA 1.6×10(-3) to 4.8×10(-3) mol dm(-3), sulfuric acid 1×10(-3) to 8×10(-3) mol dm(-3), κ-carrageenan 0.4-1.8 g dm(-3) as well as time duration 60-180 min and temperature 25-45 °C has been studied. The water swelling capacity of graft copolymer is investigated. Flocculation property for both coking and non-coking coals is studied for the treatment of coal mine waste water. The graft copolymer has been characterized by FTIR and thermogravimetric analysis.     

Physicochemical and biological characterization of SB2, a biosimilar of Remicade® (infliximab)

A biosimilar is a biological medicinal product that contains a version of the active substance of an already authorized original biological medicinal product. Biosimilarity to the reference product (RP) in terms of quality characteristics, such as physicochemical and biological properties, safety, and efficacy, based on a comprehensive comparability exercise needs to be established. SB2 (Flixabi® and Renflexis®) is a biosimilar to Remicade® (infliximab). The development of SB2 was performed in accordance with relevant guidelines of the International Conference on Harmonisation, the European Medicines Agency, and the United States Food and Drug Administration. To determine whether critical quality attributes meet quality standards, an extensive characterization test was performed with more than 80 lots of EU- and US-sourced RP. The physicochemical characterization study results revealed that SB2 was similar to the RP. Although a few differences in physicochemical attributes were observed, the evidence from the related literature, structure-activity relationship studies, and comparative biological assays showed that these differences were unlikely to be clinically meaningful. The biological characterization results showed that SB2 was similar to the RP in terms of tumor necrosis factor–α (TNF-α) binding and TNF-α neutralization activities as a main mode of action. SB2 was also similar in Fc-related biological activities including antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, neonatal Fc receptor binding, C1q binding, and Fc gamma receptor binding activities. These analytical findings support that SB2 is similar to the RP and also provide confidence of biosimilarity in terms of clinical safety and efficacy.     

Comparison of the Diversilab® system with multi-locus sequence typing and pulsed-field gel electrophoresis for the characterization of Streptococcus agalactiae invasive strains

Streptococcus agalactiae (or group B streptococcus; GBS) is a leading cause of neonatal morbidity and mortality in the developed countries. Several epidemiological typing tools have been developed for GBS to investigate the association between genotype and disease and to assess genetic variations within genogroups. This study compared the semi-automated repetitive sequence-based PCR Diversilab® system (DL) with MLST and pulsed field gel electrophoresis (PFGE) for determining the relatedness of invasive GBS strains. We analysed 179 unrelated GBS strains isolated from adult (n = 108) and neonatal (n = 71) invasive infections. By MLST, strains were resolved into 6 clonal complexes (CCs) including 23 sequence-types (STs), and 4 unique STs, whereas DL differentiated these isolates into 12 rep-PCR clusters (rPCs) and 9 unique rep-PCR types. The discriminatory power of both methods was similar, with Simpson's diversity indexes of 71.9% and 70.6%, respectively. However, their clustering concordance was low with Wallace concordance coefficients inferior to 0.4. PFGE was performed on 31 isolates representative of the most relevant DLrPCs clustered within the 3 major MLST CCs (CC-17, CC-23 and CC-1). As already observed with MLST, the concordance of DL with PFGE was low for all three CCs (Wallace coefficient < 0.5), PFGE being more discriminative than rep-PCR. In summary, this work suggests that DL is less appropriate than MLST or PFGE to study GBS population genetic diversity.     

In-depth structural characterization of Kadcyla® (ado-trastuzumab emtansine) and its biosimilar candidate

The biopharmaceutical industry has become increasingly focused on developing biosimilars as less expensive therapeutic products. As a consequence, the regulatory approval of 2 antibody-drug conjugates (ADCs), Kadcyla® and Adcetris® has led to the development of biosimilar versions by companies located worldwide. Because of the increased complexity of ADC samples that results from the heterogeneity of conjugation, it is imperative that close attention be paid to the critical quality attributes (CQAs) that stem from the conjugation process during ADC biosimilar development process. A combination of physicochemical, immunological, and biological methods are warranted in order to demonstrate the identity, purity, concentration, and activity (potency or strength) of ADC samples. As described here, we performed extensive characterization of a lysine conjugated ADC, ado-trastuzumab emtansine, and compared its CQAs between the reference product (Kadcyla®) and a candidate biosimilar. Primary amino acid sequences, drug-to-antibody ratios (DARs), conjugation sites and site occupancy data were acquired and compared by LC/MS methods. Furthermore, thermal stability, free drug content, and impurities were analyzed to further determine the comparability of the 2 ADCs. Finally, biological activities were compared between Kadcyla® and biosimilar ADCs using a cytotoxic activity assay and a HER2 binding assay. The in-depth characterization helps to establish product CQAs, and is vital for ADC biosimilars development to ensure their comparability with the reference product, as well as product safety.     

IMRT/VMAT dose distributions generated for HD® and Millennium® collimators TrueBeam® and Clinac® accelerators

AimThe aim of this study is to answer the question whether the calculated dose distributions for HD and Millennium collimators (Varian Medical Systems) are equivalent for large treatment volumes.BackgroundModern biomedical linacs are equipped with multileaf collimators where leaves can be of different widths. Thinner leaves allow better fit to desired (tumor) shape. At the same time, however, the maximum size of the field that can be obtained with the collimator is also reduced. Varian Medical Systems HD and Millennium collimators can be a good sample. They have 40 cm or 22 cm × 40 cm maximal field size at the isocenter, respectively.Materials and methodsThis paper presents the comparison of selected statistical and dosimetric parameters achieved for treatment plans where the beams for a HD collimator had to be merged because of the size of the tumor volume.Results and discussionAchieved results show that, independently from irradiated volume, there is no statistically significant difference for calculated dose distributions, integral doses, MU values and coefficients evaluating dose distributions for HD and Millennium collimators.ConclusionsResults show that both types of collimators can be used interchangeably for preparing the treatment plans for large tumor volume without quality reduction of the prepared treatment plan.     

Streamlining process characterization efforts using the high throughput ambr® crossflow system for ultrafiltration and diafiltration processing of monoclonal antibodies

Commercial process development for biopharmaceuticals often involves process characterization (PC) studies to gain process knowledge and understanding in preparation for process validation. One common approach to conduct PC activities is by using design-of-experiment, which can help determine the impact process parameter deviations may have on product quality attributes. Qualified scale-down systems are typically used to conduct these studies. For an ultrafiltration/diafiltration (UF/DF) application, however, a traditional scale-down still requires hundreds of milliliters of material per run and can only conduct one experiment at a time. This poses a challenge in resources as there could be 20+ experiments required for a typical UF/DF PC study. One solution to circumvent this is the use of high-throughput systems, which enable parallel experimentation by only using a fraction of the resources. Sartorius Stedim Biotech has recently commercialized the ambr® crossflow high-throughput system to meet this need. In this study, the performance of this system during a monoclonal antibody UF/DF step was first compared with a pilot- and a manufacturing-scale tangential flow filtration (TFF) system at a single operating condition. Due to material limitations, it was then compared to only the pilot-scale TFF system across wider ranges of transmembrane pressure; crossflow rate; and diafiltration concentration in a PC study. Permeate flux, aggregate content, process yield, pH/conductivity traces, retentate concentration, axial pressure drop, and turbidity values were measured at both scales. A good agreement was attained across scales, further supporting its potential use as a scale-down system.     

Generation and characterization of an inter-generic bivalent alpha domain fusion protein αCS from Clostridium perfringens and Staphylococcus aureus for concurrent diagnosis and therapeutic applications

Aim: To evaluate an inter‐generic recombinant alpha domain fusion protein for simultaneous detection and neutralization of Clostridium perfringens and Staphylococcus aureus alpha toxins. Methods and Results: Truncated portions of clostridial and staphylococcal alpha haemolysin genes were PCR amplified and linked to each other through a hydrophilic flexible Glycine linker sequence using overlap‐extension PCR to form a chimeric gene αCS. The recombinant αCS fusion protein was expressed and characterized for its toxicity, cell binding capacity and haemolysis inhibition properties. The fusion protein was nontoxic and effectively retarded staphylococcal alpha haemolysis, probably by competitively interacting with putative staphylococcal alpha haemolysin receptors on erythrocytes. Murine hyperimmune polysera raised against r‐αCS specifically detected 42‐kDa and 33‐kDa proteins when culture supernatants of Cl. perfringens (clostridial alpha toxin) and Staph. aureus (staphylococcal alpha toxin), respectively, were analysed in Western blot. The polyclonal antisera effectively diminished the haemolytic action of both the wild‐type toxins in vitro. Conclusions: The r‐αCS fusion protein was nontoxic competitive inhibitor of staphylococcal alpha haemolysin. The protein elicited specific immune response against Cl. perfringens and Staph. aureus alpha toxins. The antisera also neutralized the toxicities of both the native wild‐type toxins in vitro. Significance of the Study: The bivalent recombinant αCS protein could be a novel intervention in the field of diagnostics and therapeutics against Cl. perfringens and Staph. aureus infections, particularly, in case of co‐infections like gangrenous ischaemia, gangrenous mastitis, etc.     

Dosimetric and kinetic investigations of γ-irradiated sodium tartrate dihydrate

Effects of gamma radiation on solid sodium tartrate dihydrate (NaTA) were studied using electron spin resonance (ESR) spectroscopy. One main singlet located at g = 2.0034 and many weak lines located at low and high magnetic field sides were found in the irradiated samples. Dosimetric and kinetic features of the radical species responsible for the experimental ESR spectra were explored through the variations in the signal intensities with respect to applied microwave power, temperature and storage time. Activation energies of the involved radical species were also determined using data derived from annealing studies.     

Molecular characterization and identification of surrogate substrates for diacylglycerol lipase α

Diacylglycerol lipase α is the key enzyme in the formation of the most prevalent endocannabinoid, 2-arachidonoylglycerol in the brain. In this study we identified the catalytic triad of diacylglycerol lipase α, consisting of serine 472, aspartate 524 and histidine 650. A truncated version of diacylglycerol lipase α, spanning residues 1-687 retains complete catalytic activity suggesting that the C-terminal domain is not required for catalysis. We also report the discovery and the characterization of fluorogenic and chromogenic substrates for diacylglycerol lipase α. Assays performed with these substrates demonstrate equipotent inhibition of diacylglycerol lipase α by tetrahydrolipastatin and RHC-20867 as compared to reactions performed with the native diacylglycerol substrate. Thus, confirming the utility of assays using these substrates for identification and kinetic characterization of inhibitors from pharmaceutical collections.     

Preparation and characterization of biotinylated probes for the β-interferon receptor

• 1.1. Recently we described the isolation of the β-interferon receptor [Zhang et al. (1986) J. biol. Chem. 261, 8017–8021]. A highly purified product was obtained but in low quantities.
• 2.2. The use ofbiotinylated β-interferon as a ligand represents an alternate approach to receptor isolation.
• 3.3. We have prepared and characterized the derivatives N-(biotinyl)- and N-(biotinyl-ϵ-aminocaproyl)-recombinant human [Ser¹⁷-interferon β (B- and BC-recHulFNβ).
• 4.4. Biotin incorporation does not result in any loss of antiviral activity, demonstrating the recognition of the derivative by the cell receptor.
• 5.5. The biotinylated recHuIFNβ binds specifically and reversibly to succinoylavidin or guanidine thiocyanate-stripped succinoylavidin linked to a Sepharose matrix.
• 6.6. Comparison of the competition curves obtained with [¹⁴C]biotin and [³H]biotinyl recHuIFN, in the presence of increasing concentrations of biotin suggests that the IFN moiety of the derivative has little effect on the affinity of biotin for avidin.
• 7.7. Biotinylated recHuIFNβ derivatives represent useful probes for the β-IFN receptor.