Nanoparticulate Impurities in Pharmaceutical-Grade Sugars and their Interference with Light Scattering-Based Analysis of Protein Formulations.

PURPOSE: In the present study we investigated the root-cause of an interference signal (100-200 nm) of sugar-containing solutions in dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) and its consequences for the analysis of particles in biopharmaceutical drug products. METHODS: Different sugars as well as sucrose of various purity grades, suppliers and lots were analyzed by DLS and NTA before and (only for sucrose) after treatment by ultrafiltration and diafiltration. Furthermore, Fourier transform infrared (FTIR) microscopy, scanning electron microscopy coupled energy-dispersive X-ray spectroscopy (SEM-EDX), and fluorescence spectroscopy were employed. RESULTS: The intensity of the interference signal differed between sugar types, sucrose of various purity grades, suppliers, and batches of the same supplier. The interference signal could be successfully eliminated from a sucrose solution by ultrafiltration (0.02 µm pore size). Nanoparticles, apparently composed of dextrans, ash components and aromatic colorants that were not completely removed during the sugar refinement process, were found responsible for the interference and were successfully purified from sucrose solutions. CONCLUSIONS: The interference signal of sugar-containing solutions in DLS and NTA is due to the presence of nanoparticulate impurities. The nanoparticles present in sucrose were identified as agglomerates of various impurities originating from raw materials.

Pseudophosphorylated αB-crystallin is a nuclear chaperone imported into the nucleus with help of the SMN complex.

The human small heat shock protein αB-crystallin (HspB5) is a molecular chaperone which is mainly localized in the cytoplasm. A small fraction can also be found in nuclear speckles, of which the localization is mediated by successional phosphorylation at Ser-59 and Ser-45. αB-crystallin does not contain a canonical nuclear localization signal sequence and the mechanism by which αB-crystallin is imported into the nucleus is not known. Here we show that after heat shock pseudophosphorylated αB-crystallin mutant αB-STD, in which all three phosphorylatable serine residues (Ser-19, Ser-45 and Ser-59) were replaced by negatively charged aspartate residues, is released from the nuclear speckles. This allows αB-crystallin to chaperone proteins in the nucleoplasm, as shown by the ability of αB-STD to restore nuclear firefly luciferase activity after a heat shock. With the help of a yeast two-hybrid screen we found that αB-crystallin can interact with the C-terminal part of Gemin3 and confirmed this interaction by co-immunoprecipitation. Gemin3 is a component of the SMN complex, which is involved in the assembly and nuclear import of U-snRNPs. Knockdown of Gemin3 in an in situ nuclear import assay strongly reduced the accumulation of αB-STD in nuclear speckles. Furthermore, depletion of SMN inhibited nuclear import of fluorescently labeled recombinant αB-STD in an in vitro nuclear import assay, which could be restored by the addition of purified SMN complex. These results show that the SMN-complex facilitates the accumulation of hyperphosphorylated αB-crystallin in nuclear speckles, thereby creating a chaperone depot enabling a rapid chaperone function in the nucleus in response to stress.

Evaluation of sugammadex self-association.

Sugammadex, a thiolated γCD derivative used as an antagonist of steroidal blockers, was studied with regard to its tendency to self-associate in aqueous solution. Three independent methods - permeation through semi-permeable cellophane membranes, dynamic light scattering, and sedimentation equilibrium analytical ultracentrifugation - were used for this purpose. The results were in agreement with each other and showed no evidence of self-association in a wide sugammadex concentration range from 0.25 to 100mg/ml.

Strategies for the assessment of protein aggregates in pharmaceutical biotech product development.

Within the European Immunogenicity Platform (EIP) ( http://www.e-i-p.eu ), the Protein Characterization Subcommittee (EIP-PCS) has been established to discuss and exchange experience of protein characterization in relation to unwanted immunogenicity. In this commentary, we, as representatives of EIP-PCS, review the current state of methods for analysis of protein aggregates. Moreover, we elaborate on why these methods should be used during product development and make recommendations to the biotech community with regard to strategies for their application during the development of protein therapeutics.

The small heat-shock proteins HSPB2 and HSPB3 form well-defined heterooligomers in a unique 3 to 1 subunit ratio.

Various mammalian small heat-shock proteins (sHSPs) can interact with one another to form large polydisperse assemblies. In muscle cells, HSPB2/MKBP (myotonic dystrophy protein kinase-binding protein) and HSPB3 have been shown to form an independent complex. To date, the biochemical properties of this complex have not been thoroughly characterized. In this study, we show that recombinant HSPB2 and HSPB3 can be successfully purified from Escherichia coli cells co-expressing both proteins. Nanoelectrospray ionization mass spectrometry and sedimentation velocity analytical ultracentrifugation analysis showed that HSPB2/B3 forms a series of well defined hetero-oligomers, consisting of 4, 8, 12, 16, 20 and 24 subunits, each maintaining a strict 3:1 HSPB2/HSPB3 subunit ratio. These complexes are thermally stable up to 40 degrees C, as determined by far-UV circular dichroism spectroscopy. Surprisingly, HSPB2/B3 exerted a poor chaperone-like and thermoprotective activity, which is likely related to the low surface hydrophobicity, as revealed by its interaction with the hydrophobic probe 1-anilino-8-naphthalenesulfonic acid. Co-immunoprecipitation experiments demonstrated that the HSPB2/B3 oligomer cannot interact with HSP20, HSP27 or alphaB-crystallin, whereas the homomeric form of HSPB2, thus not in complex with HSPB3, could associate efficiently with HSP20. Taken altogether, this study provides evidence that, despite the high level of sequence homology within the sHSP family the biochemical properties of the HSPB2/B3 complex are distinctly different from those of other sHSPs, indicating that the HSPB2/B3 assembly is likely to possess cellular functions other than those of its family members.

Nuclear import of {alpha}B-crystallin is phosphorylation-dependent and hampered by hyperphosphorylation of the myopathy-related mutant R120G.

Phosphorylation modulates the functioning of alphaB-crystallin as a molecular chaperone. We here explore the role of phosphorylation in the nuclear import and cellular localization of alphaB-crystallin in HeLa cells. Inhibition of nuclear export demonstrated that phosphorylation of alphaB-crystallin is required for import into the nucleus. As revealed by mutant analysis, phosphorylation at Ser-59 is crucial for nuclear import, and phosphorylation at Ser-45 is required for speckle localization. Co-immunoprecipitation experiments suggested that the import of alphaB-crystallin is possibly regulated by its phosphorylation-dependent interaction with the survival motor neuron (SMN) protein, an important factor in small nuclear ribonucleoprotein nuclear import and assembly. This interaction was supported by co-localization of endogenous phosphorylated alphaB-crystallin with SMN in nuclear structures. The cardiomyopathy-causing alphaB-crystallin mutant R120G was found to be excessively phosphorylated, which disturbed SMN interaction and nuclear import, and resulted in the formation of cytoplasmic inclusions. Like for other protein aggregation disorders, hyperphosphorylation appears as an important aspect of the pathogenicity of alphaB-crystallin R120G.

Mimicking phosphorylation of the small heat-shock protein alphaB-crystallin recruits the F-box protein FBX4 to nuclear SC35 speckles.

The mammalian small heat shock protein alphaB-crystallin can be phosphorylated at three different sites, Ser19, Ser45 and Ser59. We compared the intracellular distribution of wild-type, nonphosphorylatable and all possible pseudophosphorylation mutants of alphaB-crystallin by immunoblot and immunocytochemical analyses of stable and transiently transfected cells. We observed that pseudophosphorylation at two (especially S19D/S45D) or all three (S19D/S45D/S59D) sites induced the partial translocation of alphaB-crystallin from the detergent-soluble to the detergent-insoluble fraction. Double immunofluorescence studies showed that the pseudophosphorylation mutants localized in nuclear speckles containing the splicing factor SC35. The alphaB-crystallin mutants in these speckles were resistant to mild detergent treatment, and also to DNase I or RNase A digestion, indicating a stable interaction with one or more speckle proteins, not dependent on intact DNA or RNA. We further found that FBX4, an adaptor protein of the ubiquitin-protein isopeptide ligase SKP1/CUL1/F-box known to interact with pseudophosphorylated alphaB-crystallin, was also recruited to SC35 speckles when cotransfected with the pseudophosphorylation mutants. Because SC35 speckles also react with an antibody against alphaB-crystallin endogenously phosphorylated at Ser45, our findings suggest that alphaB-crystallin has a phosphorylation-dependent role in the ubiquitination of a component of SC35 speckles.

The small heat-shock protein alpha B-crystallin promotes FBX4-dependent ubiquitination.

AlphaB-crystallin is a small heat-shock protein in which three serine residues (positions 19, 45, and 59) can be phosphorylated under various conditions. We describe here the interaction of alphaB-crystallin with FBX4, an F-box-containing protein that is a component of the ubiquitin-protein isopeptide ligase SCF (SKP1/CUL1/F-box). The interaction with FBX4 was enhanced by mimicking phosphorylation of alphaB-crystallin at both Ser-19 and Ser-45 (S19D/S45D), but not at other combinations. Ser-19 and Ser-45 are preferentially phosphorylated during the mitotic phase of the cell cycle. Also alphaB-crystallin R120G, a mutant found to co-segregate with a desmin-related myopathy, displayed increased interaction with FBX4. Both alphaB-crystallin S19D/S45D and R120G specifically translocated FBX4 to the detergent-insoluble fraction and stimulated the ubiquitination of one or a few yet unknown proteins. These findings implicate the involvement of alphaB-crystallin in the ubiquitin/proteasome pathway in a phosphorylation- and cell cycle-dependent manner and may provide new insights into the alphaB-crystallin-induced aggregation in desmin-related myopathy.