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Aggressive Light Chain Amyloidosis Cases have Unstable Immunoglobulin Light Chains
Published in Gilles Grateau, Robert A. Kyle, Martha Skinner, Amyloid and Amyloidosis, 2004
L. A. Sikkink, E. M. Baden, S. R. Vance, K. J.-L. Riley, A.T. Moch, M. Ramirez-Alvarado
cDNAs from patients’ bone marrow were used to clone the protein genes in E. coli expression vectors except uMM-01 that was purified from a urine sample from the patient. Proteins were purified using size exclusion chromatography. Overall secondary structure was determined by Far UV Circular Dichroism spectroscopy (CD). Protein stability analysis was followed by thermal and chemical denaturation followed by CD or Fluorescence spectroscopy.
Proteins in Cosmetics
Published in E. Desmond Goddard, James V. Gruber, Principles of Polymer Science and Technology in Cosmetics and Personal Care, 1999
E. Desmond Goddard, James V. Gruber
Size exclusion chromatography (also referred to as gel filtration and gel permeation chromatography) is the most practical and frequently employed technique to fractionate polypeptides in function of their dimension and provides the basis to estimate the average molecular weight of protein blends. Exclusion columns contain porous particles of a hydrophilic polymer with different pore diameters. When a polypeptide mixture is forced to pass through the column, its components diffuse into those pores that have a diameter greater than their effective diameters (the effective diameter will include any molecules of solvation). If a solute is of such a diameter that it cannot diffuse into any of the pores, it will be unretained and elute in the void volume of the column. Conversely, a polypeptide that is capable of diffusing completely into all of the pores will totally permeate the packing and require a much larger volume of solvent to achieve elution. The separation of molecules having different diameters is based on the extent of their diffusion through the pores and the different time needed to achieve their elution. An ideal size exclusion fractionation will occur when all solutes diffuse into part of the pores of the stationary phase. Size exclusion chromatography is a way of rapidly obtaining an estimate of the molecular weight of a sample, by comparison with authentic standards. To correlate the elution time of the polypeptides (which is a function of their size) with their molecular weight, the solutes and standards should have the same spatial conformation. For blends of molecules having homogeneous shape, over a considerable range, the elution volume is approximately a linear function of the logarithm of molecular weight. Practically, molecular masses of polypeptides are only approximately determinable as their hydrodynamic
PEGylated Dendritic Nanoparticulate Carriers of Anti-Cancer Drugs
Published in Mansoor M. Amiji, Nanotechnology for Cancer Therapy, 2006
D. Bhadra, S. Bhadra, N. K. Jain
The dendrimers that are most studied and reported up to higher generations (Bosman, Janssen, and Meijer 1999) can be classified chemically viz. peptide dendrimers that were patented and described upto higher generations by Denkewalter in the early 1980s. Those can well be characterized by size-exclusion chromatography only. PAMAM dendrimers are another class of dendritic structures that have been thoroughly investigated and had received wide spread attention. These were divergently constructed, e.g., PAMAM described by Tomalia and arborol systems of Newkome. Polypropyleneimine dendrimers were produced by Vogtle by divergent synthesis. It was also produced by heterogeneously catalyzed hydrogenation by De Brabander-van-den Berg and Meijer (1993) and is also studied widely. Frechet produced Poly-ether dendrimers by convergent procedures (Wooley et al. 1994). These also include Poly(aryl ether) dendrimers. Moore produced Phenyl acetylene dendrimers by convergent approach. Tetrathiofulvalene (TTF)21-glycol dendrimers are another class of dendrimers that are intra-dendrimer aggregates of TTF cation radicals (Christensen et al. 1998). These are evident by spectrochemical studies of partially oxidized TTF units. These were prepared by convergent approaches. Pentaporphyrin is another starburst porphyrin polymer that is considered as a first generation dendrimers (Norsten and Branda 1998). These had hybrid properties of porphyrin and dendrimers where ether linkages are found for iterations. Aryl ester monodispersed dendrimers based on 1, 3, 5-benzene tricarboxylic acid were initially described by Miller, Kwock, and Neenan (1992) and prepared by convergent synthesis. This was possibly used for MWs standards as polymeric rheological modifier, molecular inclusion hosts, etc. Two types of dendrimeric nucleic acids have been studied, viz. those that are covalently connected, and those that self-assemble via complementary base-pairing schemes (Kim and Zimmerman 1998). An example of a covalent DNA dendrimer was reported, wherein a second-generation phosphodiester-based dendrimer derived from pentaerythritol held nine pentathymidylate units on the end-groups and either a 15-mer or 26-mer at the core.
High concentration formulation developability approaches and considerations
Published in mAbs, 2023
Jonathan Zarzar, Tarik Khan, Maniraj Bhagawati, Benjamin Weiche, Jasmin Sydow-Andersen, Alavattam Sreedhara
Regardless of the study design, detailed characterization of the observed impurities/degradation products (prominently high molecular weight species and/or particles) is required. This endeavor can be challenging and requires the use of a specialized toolkit of analytical methods. Apart from the low-resolution biophysical methods used for investigation of protein oligomerization and complex formation, such as DLS or more time-consuming experimental techniques like analytical ultracentrifugation, the most established and representative method for assessment of species of different molecular weights in the biopharmaceutical industry is size exclusion chromatography (SEC). While SEC alone does not provide an accurate size of the observed species, coupling it to a multi-angle light scattering (MALS) detector enables determination of the molar mass and concentration, which are proportional to the light scattered by the particles.52 Despite the wide and validated applicability of the SEC method for release and stability monitoring, further investigation of aggregation phenomena may require orthogonal techniques to SEC to gain additional or more precise data that is typically not necessary for release and stability monitoring.
An adapted consensus protein design strategy for identifying globally optimal biotherapeutics
Published in mAbs, 2022
Yanyun Liu, Kenny Tsang, Michelle Mays, Gale Hansen, Jeffrey Chiecko, Maureen Crames, Yangjie Wei, Weijie Zhou, Chase Fredrick, James Hu, Dongmei Liu, Douglas Gebhard, Zhong-Fu Huang, Akshita Datar, Anthony Kronkaitis, Kristina Gueneva-Boucheva, Daniel Seeliger, Fei Han, Saurabh Sen, Srinath Kasturirangan, Justin M. Scheer, Andrew E. Nixon, Tadas Panavas, Michael S. Marlow, Sandeep Kumar
Protein samples (at 10 g/L) were dispensed into screw-cap polypropylene microcentrifuge tubes and placed in an incubator set at 40°C. Samples were extracted on d 0, 7, 21, and 35 and diluted to 1 g/L with buffer. Analytical size exclusion chromatography (SEC) was carried out using the same Agilent HPLC system as described for analysis of stability samples, above. Ten micrograms of samples were injected onto two tandemly connected Acquity BEH200 columns (Waters, 1.7 µm, 4.6 × 150 mm) at a flow rate of 0.25 mL/min. The mobile phase contains 50 mM sodium phosphate, 200 mM arginine chloride, 0.05% sodium azide, pH 6.8. The elution profile was monitored at 280 nm and manually integrated to calculate the level of HMW and LMW species. Both HMW and LMW species were plotted as a function of storage time and then fit by linear regression to derive the slope to reflect the degradation kinetics. R2 was used to check the quality of the linear fit.
Unraveling the complexity of the extracellular vesicle landscape with advanced proteomics
Published in Expert Review of Proteomics, 2022
Julia Morales-Sanfrutos, Javier Munoz
EVs can be separated from soluble proteins and protein aggregates by density gradient ultracentrifugation (DG), since vesicles have lower densities (1.13–1.19 g/mL) than proteins (1.35–1.41 g/mL). In this strategy, a continuous or discontinuous pre-constructed density gradient, which increases in density from top to bottom, is used. Different mediums are available, being sucrose and iodixanol (Optiprep) most commonly used. Upon ultracentrifugation for several hours, sEVs (including exosomes) migrate to the point where their density is the same as the medium density. DG is very effective in separating sEVs from proteins and non-membranous contaminations, avoiding vesicle aggregation, and can even separate EVs subtypes. However, these benefits are accompanied by increased costs, time and workload. In addition, this strategy requires an extra step, such as ultracentrifugation or ultrafiltration, to remove the density medium prior to MS analysis. In combination with dUC, DG was one of the first attempts to address the heterogeneity of EVs [18].(3) Size-exclusion chromatography