How subvisible particles become invisible-relevance of the refractive index for protein particle analysis.

The aim of the present study was to quantitatively assess the relevance of transparency and refractive index (RI) on protein particle analysis by the light-based techniques light obscuration (LO) and Micro-Flow Imaging (MFI). A novel method for determining the RI of protein particles was developed and provided an RI of 1.41 for protein particles from two different proteins. An increased RI of the formulation by high protein concentration and/or sugars at pharmaceutically relevant levels was shown to lead to a significant underestimation of the subvisible particle concentration determined by LO and MFI. An RI match even caused particles to become "invisible" for the system, that is, not detectable anymore by LO and MFI. To determine the influence of formulation RI on particle measurements, we suggest the use of polytetrafluoroethylene (PTFE) particles to test a specific formulation for RI effects. In case of RI influences, we recommend also using a light-independent technique such as resonant mass measurement (RMM) (Archimedes) for subvisible particle analysis in protein formulations.

Particles in therapeutic protein formulations, Part 1: overview of analytical methods.

The presence of particles is a major issue during therapeutic protein formulation development. Both proteinaceous and nonproteinaceous particles need to be analyzed not only due to the requirements of the Pharmacopeias but also to monitor the stability of the protein formulation. Increasing concerns about the immunogenic potential together with new developments in particle analysis make a comparative description of established and novel analytical methods useful. Our review aims to provide a comprehensive overview on analytical methods for the detection and characterization of visible and subvisible particles in therapeutic protein formulations. We describe the underlying theory, benefits, shortcomings, and illustrative examples for quantification techniques, as well as characterization techniques for particle shape, morphology, structure, and identity.

New method to produce equine antirabies immunoglobulin F(ab')2 fragments from crude plasma in high quality and yield.

Rabies is still a major cause of human deaths in several developing countries. According to the World Health Organization, administration of antirabies serum or antirabies immunoglobulin is recommended for patients who have experienced a category-III exposure to rabies. Improvement of antirabies immunoglobulin production is required to enhance safety and efficacy of the products. In this paper, a new method to produce equine antirabies immunoglobulin F(ab')(2) fragments from crude plasma is proposed. First, protein G affinity chromatography was used to purify IgG from equine plasma. Moreover, purification of IgG was shown to facilitate its digestion by pepsin. Compared to the direct digestion of crude plasma, a lower amount of pepsin and a shorter digestion time were required to completely digest the purified IgG to F(ab')(2). Complete digestion of purified IgG to F(ab')(2) was achieved at a pepsin/IgG (w/w) ratio of 5:45 with preservation of structure and potency. Finally, purification of F(ab')(2) was accomplished by a combination of protein A affinity chromatography and ultrafiltration with a 50-kDa nominal molecular weight cut-off membrane. The new process resulted in 68.9±0.6 (%) total recovery of F(ab')(2) and a F(ab')(2) product of high potency.

Plain and mono-pegylated recombinant human insulin exhibit similar stress-induced aggregation profiles.

PEGylation has been suggested to improve the stability of insulin, but evidence for that is scarce. Here, we compared the forced aggregation behavior of insulin and mono-PEGylated insulin. Therefore, recombinant human insulin was conjugated on lysine B29 with 5-kDa PEG. PEG-insulin was purified by size-exclusion chromatography (SEC) and characterized by mass spectrometry (MS). Next, insulin and PEG-insulin were subjected to heating at 75 °C, metal-catalyzed oxidation, and glutaraldehyde cross-linking. The products were characterized physicochemically by complementary analytical methods. Mono-PEGylation of insulin was confirmed by SEC and MS. Under each of the applied stress conditions, insulin and PEG-insulin showed comparable degradation profiles. All the stressed samples showed submicron aggregates in the size range between 50 and 500 nm. Covalent aggregates and conformational changes were found for both oxidized products. Insulin and its PEGylated counterpart also exhibited similar characteristics when exposed to heat stress, that is, slightly changed secondary and tertiary structures, covalent aggregates with partially intact epitopes, and separation of chain A from chain B. Both glutaraldehyde-treated insulin and PEG-insulin contained covalent and noncovalent aggregates with intact epitopes, showed partially perturbed secondary structure, and substantial loss of tertiary structure. From these results, we conclude that PEGylation does not protect insulin against forced aggregation.

The molecular chaperone alpha-crystallin as an excipient in an insulin formulation.

PURPOSE: To investigate insulin fibrillation under accelerated stress conditions in the presence of a novel excipient, the molecular chaperone alpha-crystallin, in comparison with common excipients. METHODS: To induce fibrillation, recombinant human insulin (0.58 mg ml(-1)) formulations without excipient or with bovine alpha-crystallin (0.01-0.2 mg ml(-1)), human serum albumin (1-5 mg ml(-1)), sucrose (10-100 mg ml(-1)) or polysorbate 80 (0.075-0.3 mg ml(-1)) were subjected to stirring stress in a fluorescence well plate reader and formulation vials. Protein fibrillation was monitored by thioflavin T. The formulations were further characterized by size-exclusion chromatography, light obscuration, UV/Vis and circular dichroism spectroscopy. RESULTS: In both methods, insulin formed thioflavin T-binding species, most likely fibrils. Addition of alpha-crystallin in the well plate assay greatly improved insulin's resistance to fibrillation, measured as a 6-fold increase in fibrillation lag time for the lowest and 26-fold for the highest concentration used, whereas all other excipients showed only a marginal increase in lag time. The stabilizing effect of alpha-crystallin was shown by all characterization techniques used. CONCLUSIONS: The effect of alpha-crystallin on insulin's physical stability outperforms that of commonly used excipients. alpha-Crystallin is proposed to bind specifically to pre-fibrillation species, thereby inhibiting fibrillation. This makes alpha-crystallin an interesting excipient for proteins with propensity to fibrillate.

Elution behavior of insulin on high-performance size exclusion chromatography at neutral pH.

The pharmacopoeia protocol for HP-SEC of insulin, using an acidic non-physiological eluent, does not represent insulin's association state in the formulation. This study aimed to evaluate insulin's elution behavior in HP-SEC in a "physiological" (aqueous, neutral pH) eluent, using on-line UV absorption and multi-angle laser light scattering detection. The effect of insulin concentration and association state in the formulation (monitored by circular dichroism) and eluent composition (zinc ion, arginine) on its elution behavior was assessed. We showed that the elution behavior of insulin in "physiological" HP-SEC is affected by both dynamic association-dissociation of insulin molecules and insulin-column interactions. Insulin molecules re-equilibrated in the HP-SEC eluent, making its elution behavior practically insensitive to the association state of insulin in the formulation. Zinc ions in the eluent promoted association of insulin to hexamers, whereas arginine overruled the effect of zinc ions and induced on-column dissociation of insulin to dimers and monomers. Combined results from "physiological" and compendial HP-SEC were shown to provide a better view of the aggregation state of heat-stressed insulin than either of the single methods. The insights obtained with this study are crucial for a proper evaluation of HP-SEC data of insulin.

Swelling lecithin: cholesterol implants for the controlled release of proteins.

This work demonstrated the effect of two salts as potential simple formulation excipients in modifying hydration properties, phase behavior, and protein release from lecithin-based implants. In vitro release of a model protein, bovine serum albumin (BSA), from cylindrical-shaped lecithin and lecithin:cholesterol (1:1 w/w) implants containing 0, 10, or 30% w/w NaCl or CaCl2 was studied. In the absence of salts, BSA was released from lecithin and lecithin:cholesterol implants with a high monomer content and the release profiles were similar to those previously reported. Cholesterol increased the swelling, induced the formation of myelin structures, and reduced BSA release from the matrices. Addition of the salts to lecithin:cholesterol implants further enhanced the swelling, altered the hydrated morphology, and inhibited protein release. Analyses showed that BSA associated into multimers within these swollen lipid matrices but retained a high degree of protein native structure. Factors that may have contributed to the inhibition of the in vitro release included 1) the swollen multilamellar layers assembled as diffusional barriers, 2) adsorption of BSA onto the hydrated lipid vesicles, and 3) formation of protein aggregates.