Aggregation of human plasma and of human blood induced in vitro by pegfilgrastim originator formulation buffer and pegfilgrastim products.

PEGylated recombinant human granulocyte colony stimulating factor (pegfilgrastim) is used clinically to reduce the incidence and duration of severe neutropenia in patients who have received chemotherapy treatment. Pegfilgrastim products are administered by subcutaneous injection. We herein report that solutions of pegfilgrastim originator product Neulasta®, of a biosimilar product candidate, and also of the pegfilgrastim originator formulation buffer, induced aggregate formation when mixed in vitro with human plasma, and formation of large membranous aggregated structures when mixed with human blood.

Aggregation of human plasma and of human blood induced in vitro by filgrastim originator product; effect of PEGylation.

We herein report that filgrastim product Neupogen® and the filgrastim formulation buffer induced aggregate formation when mixed in vitro with human plasma, and formation of large membranous erythrocyte aggregates when mixed with human blood, similar to the aggregation induced by pegfilgrastim and by pegfilgrastim buffer [T. Arvinte, E. Poirier, N.

Part 2: Physicochemical characterization of bevacizumab in 2 mg/mL antibody solutions as used in human i.v. administration: Comparison of originator with a biosimilar candidate.

The physicochemical properties of Avastin® manufactured in the USA (Originator USA) and in Europe (Originator EU) and ABX-BEV, a bevacizumab biosimilar drug product candidate produced by Apobiologix Inc., were characterized at a clinically relevant concentration of 2 mg/mL following dilution of the 25 mg/mL drug products with 0.9% NaCl.

Part 1: Physicochemical characterization of bevacizumab in undiluted 25 mg/mL drug product solutions: Comparison of originator with a biosimilar candidate.

The biosimilarity assessment of the physicochemical properties of high-concentration biopharmaceuticals is usually performed with measurements on diluted solutions, at concentrations below 1 mg/mL. In this study 13 orthogonal, spectroscopy and particle size determination methods were used to characterize the structure and aggregation of undiluted, 25 mg/mL bevacizumab drug products Avastin® manufactured in the USA and in Europe, and ABX-BEV, a bevacizumab biosimilar candidate produced by Apobiologix Inc. Secondary structure, conformation and the potential occurrence of chemical degradation of the monoclonal antibodies were characterized and compared using infrared spectroscopy, intrinsic fluorescence and ANS fluorescence spectroscopy.

A new approach for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G.

Bispecific antibodies combine two different antigen-binding sites in a single molecule, enabling more specific targeting, novel mechanisms of action, and higher clinical efficacies. Although they have the potential to outperform conventional monoclonal antibodies, many bispecific antibodies have issues regarding production, stability, and pharmacokinetic properties. Here, we describe a new approach for generating bispecific antibodies using a common light chain format and exploiting the stable architecture of human immunoglobulin G We used iterative experimental validation and computational modeling to identify multiple Fc variant pairs that drive efficient heterodimerization of the antibody heavy chains.

Influence of protein formulation and carrier solution on asymmetrical flow field-flow fractionation: a case study of the plant-produced recombinant anthrax protective antigen pp-PA83.

Asymmetrical flow field-flow fractionation (afFFF) was used to investigate the properties of a plant-produced anthrax toxin protective antigen, pp-PA83. The afFFF fractogram consisted of two main peaks with molar masses similar to the molecular mass of pp-PA83 monomer. afFFF carrier solutions strongly influenced the ratio and the intensity of the two main peaks.

Influence of methionine oxidation on the aggregation of recombinant human growth hormone.

Oxidation of methionine (Met) residues is one of the major chemical degradations of therapeutic proteins. This chemical degradation can occur at various stages during production and storage of a biotherapeutic drug. During the oxidation process, the side chain of methionine residue undergoes a chemical modification, with the thioether group substituted by a sulfoxide group.

Aggregation of biopharmaceuticals in human plasma and human serum: implications for drug research and development.

Analytical methods based on light microscopy, 90° light-scattering and surface plasmon resonance (SPR) allowed the characterization of aggregation that can occur when antibodies are mixed with human plasma. Light microscopy showed that aggregates formed when human plasma was mixed with 5% dextrose solutions of Herceptin(®) (trastuzumab) or Avastin(®) (bevacizumab) but not Remicade(®) (infliximab). The aggregates in the plasma-Herceptin(®)-5% dextrose solution were globular, size range 0.

Validation of an automated method for compounding monoclonal antibody patient doses: case studies of Avastin (bevacizumab), Remicade (infliximab) and Herceptin (trastuzumab).

Automation robots have recently come to the market as an alternative for manual compounding of drugs for intravenous administration. Our aim was to assess whether robotic compounding can be performed with monoclonal antibodies (mAbs) without influencing the aggregation state of the proteins. Three frequently used mAbs were studied: infliximab (Remicade, Janssen Biotech) and trastuzumab (Herceptin, Roche) in lyophilised form, and bevacizumab (Avastin, Roche) as a liquid formulation stored at 2°C to 8°C.

Noncovalent PEGylation: different effects of dansyl-, L-tryptophan-, phenylbutylamino-, benzyl- and cholesteryl-PEGs on the aggregation of salmon calcitonin and lysozyme.

Protein aggregation is a major instability that can occur during all stages of protein drug production and development. Protein aggregates may compromise the safety and efficacy of the final protein formulation. In this paper, various new excipients [phenylbutylamino-, benzyl-, and cholesteryl-polyethylene glycols (PEGs)] and their use for the reduction of aggregation of salmon calcitonin (sCT) and hen egg-white lysozyme (HEWL) by noncovalent PEGylation are presented.

Ultraviolet Resonance Raman spectroscopy used to study formulations of salmon calcitonin, a starch-peptide conjugate and TGF-β3.

Ultraviolet Resonance Raman (UVRR) spectroscopy with excitation at 244 nm was investigated here as a possible useful tool for fast characterization of biopharmaceuticals. Studies were performed on three protein drugs: salmon calcitonin (sCT), starch-peptide conjugate, and transforming growth factor-β3 (TGF-β3) adsorbed onto solid granules of tricalcium phosphate (TCP). Secondary structure of sCT was investigated for solutions of 0.

Stability of seasonal influenza vaccines investigated by spectroscopy and microscopy methods.

The stability of different seasonal influenza vaccines was investigated by spectroscopy and microscopy methods before and after the following stress-conditions: (i) 2 and 4 weeks storage at 25°C, (ii) 1 day storage at 37°C and (iii) one freeze-thaw cycle. The subunit vaccine Influvac (Solvay Pharma) and the split vaccine Mutagrip (Sanofi Pasteur) were affected by all stresses. The split vaccine Fluarix (GlaxoSmithKline) was affected only by storage at 25°C.

Tryptophan-mPEGs: novel excipients that stabilize salmon calcitonin against aggregation by non-covalent PEGylation.

Protein aggregation, which is triggered by various factors, is still one of the most prevalent problems encountered during all stages of protein formulation development. In this publication, we present novel excipients, tryptophan-mPEGs (Trp-mPEGs) of 2 and 5 kDa molecular weight and suggest their use in protein formulation. The synthesis and physico-chemical characterization of the excipients are described.

Addressing new analytical challenges in protein formulation development.

As the share of therapeutic proteins in the arsenal of modern medicine continue increasing, relatively little progress has been made in the development of analytical methods that would address specific needs encountered during the development of these new drugs. Consequently, the researchers resort to adaptation of existing instrumentation to meet the demands of rigorous bioprocess and formulation development. In this report, we present a number of such adaptations as well as new instruments that allow efficient and precise measurement of critical parameters throughout the development stage.

Noncovalent pegylation by dansyl-poly(ethylene glycol)s as a new means against aggregation of salmon calcitonin.

During all stages of protein drug development, aggregation is one of the most often encountered problems. Covalent conjugation of poly(ethylene glycol) (PEG), also called PEGylation, to proteins has been shown to reduce aggregation of proteins. In this paper, new excipients based on PEG are presented that are able to reduce aggregation of salmon calcitonin (sCT).

Enhanced physical stability of human calcitonin after methionine oxidation.

Calcitonin is a blood-calcium-lowering peptide, present in different species, which inhibits the resorption of bone by osteoclasts. Human calcitonin (hCT) is one of the few calcitonin peptides, which contains a methionine residue; this residue is in position 8. Methionines are known to be readily oxidized to sulfoxides both in vivo and in vitro.

Stability of human growth hormone: influence of methionine oxidation on thermal folding.

Oxidation, particularly of methionine residues, is one of the major chemical degradations of proteins. In a previous publication we studied the conformation of recombinant human growth hormone (r-hGH) selectively oxidized at Met14 and Met125. Conformation of oxidized r-hGH was found not different from that of nonoxidized r-hGH.

Oxidized recombinant human growth hormone that maintains conformational integrity.

Chemical degradations often induce changes in protein conformation and thus influence protein activity and protein stability in solutions. One difficulty in studying of chemical degradations on protein aqueous properties is to obtain sufficient amount of chemically degraded protein which is well characterized. Chemical degradation protocols that are often used may induce also conformation changes and aggregation of the protein.

Physical instability, aggregation and conformational changes of recombinant human bone morphogenetic protein-2 (rhBMP-2).

The influence of two different pH values on the physical stability of recombinant human bone morphogenetic protein-2 (rhBMP-2) in aqueous solution was evaluated in the present work. RhBMP-2 in solution at pH 4.5 or 6.

New methods allowing the detection of protein aggregates: a case study on trastuzumab.

Aggregation compromises the safety and efficacy of therapeutic proteins. According to the manufacturer, the therapeutic immunoglobulin trastuzumab (Herceptin) should be diluted in 0.9% sodium chloride before administration.

Toward the understanding of the photodynamic activity of m-THPP encapsulated in PLGA nanoparticles: correlation between nanoparticle properties and in vivo activity.

Biodegradable polymeric nanoparticles (NP) are promising delivery systems for photosensitizers (PS). A hydrophobic PS, the meso-tetra(p-hydroxyphenyl)porphyrin (m-THPP) was encapsulated into NP made of poly(D,L-lactide-co-glycolide) using an emulsification-diffusion method, and poly(vinyl alcohol) (PVAL) as stabilizing agent during the emulsification step. Three batches of NP with mean diameters of 117, 285, and 593 nm, respectively, were prepared.

High-throughput formulation screening of therapeutic proteins.

High-throughput screening (HTS) is used extensively in drug discovery to identify active compounds. Automated preparation and sample analysis in multiwell plates using a combination of liquid and/or powder handling stations, robotics and sensitive detection devices provide powerful tools. At present, protein formulation remains a slow process and will benefit from a fast formulation screening approach.

A high throughput protein formulation platform: case study of salmon calcitonin.

Purpose: The feasibility of using high throughput spectroscopy for characterization and selection of physically stable protein formulations was studied.

Materials And Methods: A hundred aqueous formulations of salmon calcitonin (sCT) were prepared using 20 buffer compositions. The solutions had pH values between 2.

Modulation of the Bioactive Conformation of Transforming Growth Factor β: Possible Implications of Cation Binding for Biological Function.

In any organism, very precisely adjusted interaction and exchange of information between cellsis continuously required. These cooperative interactions involve numerous cytokines, acting throughcorresponding sets of cell-surface receptors. The transforming growth factor β (TGF-β)superfamily includes a variety of structurally related multifunctional cytokines that play criticalroles in maintaining cellular homeostasis and controlling cell fate.

High throughput methods to characterize protein permeation and release.

Spectroscopic methods have been developed to study protein permeation and release kinetics in multi-well plates. The permeation of bovine serum albumin (BSA) through a membrane, which separated a 96-well plate in two compartments, was characterized. A change in fluorescence intensity was measured corresponding to the permeation of BSA from one compartment to another.