In situ determination of adsorption kinetics of proteins in a finite bath

A method for fast in situ measurement of adsorption kinetics based on a finite bath was developed. We modified the conventional finite bath by replacing the external loop by a dip probe which enables in situ measurement of the concentration change in the contactor. Deposition of adsorbent particles on the reflection surface of the dip probe compromised measurements. Different membranes, a polyamide, a polypropylene and a nylon membrane were tested to protect the internal reflection surface of the dip probe from fouling with adsorbent particles. The nylon membrane provided efficient protection and high mass transfer evaluated by response time experiments. Unspecific adsorption of the model protein on the membrane could also be excluded. To corroborate the measurements of the dip probe the results were compared to a conventional finite bath and to a shallow-bed. The uptake curves for human polyclonal IgG at different concentrationes (0.1-3 g/l) on rProtein A Sepharose FF and MabSelect were used as model system. The effective diffusion coefficients were determined using a pore diffusion model. These values were in good agreement for all methods.     

Fluorescence resonance energy transfer in doubly-quantum dot labeled IgG system

The mouse immunoglobulin G (mouse IgG) as a kind of bio-molecule was labeled with two different luminescent colloidal semiconductor quantum dots (QDs), green-emitting CdTe quantum dots and red-emitting CdTe quantum dots in this work. As a result of the fluorescence resonance energy transfer (FRET) between the two different sizes nanoparticles with mouse IgG as the binding bridge, a significant enhancement of the emission of the red-emitting CdTe quantum dots and the corresponding quenching of the emission of green-emitting CdTe quantum dots were observed. The relationship between the concentration of the mouse immunoglobulin G and the fluorescence intensity ratio (I_a/I_d) of acceptors and donors was studied also. Under optimal conditions, the calibration graph is linear over the range of 0.1–20.0 mg/L mouse IgG.     

Blood protein purification and simultaneous removal of nonenveloped viruses using tangential-flow preparative electrophoresis

Gradiflow is new technology allowing purification of important blood proteins from viral contaminated plasma. Protein purification is based on unique scalable tangential-flow preparative electrophoresis, and is distinct from current technology because protein purification and virus removal are performed in the same step. This one-step removal and purification exploits both the size and charge of target proteins. The medically important blood proteins, immunoglobulin G (IgG) and alpha-1-antitrypsin, were chosen to demonstrate the ability of this process to purify proteins from contaminated plasma. Clearance factors achieved by infectivity assays and polymerase chain reaction (PCR) that meet regulatory requirements demonstrated removal of canine parvovirus (CPV). CPV is a model virus for pathogenic nonenveloped viruses, including parvovirus B19, not adequately removed or inactivated by most processes currently in practice. The recovery of proteins from plasma with high purity, recovery, and function, while simultaneously removing viruses, provides blood products with a level of purity compatible with clinical use more quickly and cheaply than available techniques.     

Simultaneous determination of albumin and immunoglobulin G with fluorescence spectroscopy and multivariate calibration

A method is proposed for the simultaneous determination of albumin and immunoglobulin G (IgG1) with fluorescence spectroscopy and multivariate calibration with partial least squares regression (PLS). The influence of some instrumental parameters were investigated with two experimental designs comprising 19 and 11 experiments, respectively. The investigated parameters were excitation and emission slit, detection voltage and scan rate. When a suitable instrumental setting had been found, a minor calibration and test set were analysed and evaluated. Thereafter, a larger calibration of albumin and IgG1 was made out of 26 samples (0-42 μg ml⁻¹ albumin and 0-12.7 μg ml⁻¹ IgG1). This calibration was validated with a test set consisting of 14 samples in the same concentration range. The precision of the method was estimated by analysing two test set samples for six times each. The scan modes tested were emission scan and synchronous scan Δ60 nm. The results showed that the method could be used for determination of albumin and IgG1 (albumin, root mean square error of prediction (RMSEP) <2, relative standard error of prediction (RSEP) <6% and IgG1, RMSEP <1, RSEP <8%) in spite of the overlapping fluorescence of the two compounds. The estimated precision was relative standard deviation (R.S.D.) <1.7%. The method was finally applied for the analysis of some sample fractions from an albumin standard used in affinity chromatography.     

Contamination of therapeutic human immunoglobulin preparations with apolipoprotein H (β2‐glycoprotein I)

Polyclonal immunoglobulin (Ig) concentrates are important biological medicinal products and the assurance of their quality and safety is crucial. In our present approach we used proteomic methods to check the purity of commercial Ig products of different origin. The experimental setup included nonreducing 2DE or DIGE combined with MALDI‐TOF and the thrombin generation assay, a routine safety test for pharmaceutical Ig preparations, and was complemented by a specific immunoassay. 2DE patterns displayed contaminations with trace amounts of human apolipoprotein H (Apo‐H), transferrin, albumin, and its fragments. In contrast to the latter, Apo‐H is a protein that is active in the coagulation cascade, and thus a potential involvement in thromboembolic events in vivo cannot be excluded. It was found by 2DE and MALDI‐TOF to be a contaminant of several Ig preparations. Spiking experiments of Ig preparations with pure Apo‐H demonstrated an Apo‐H concentration dependent increase in thrombin generation assay values. Traces of Apo‐H are possibly also contributing to unwanted side effects, as already known for factor XIa. The significance of Apo‐H contaminations for these side effects might be verified by detailed analyses of pharmacovigilance data.     

In‐Tandem Column Arrangement for Rapid Purification of IgG using a Blue Avid Gel P and an Aza‐Arenophilic Affinity Gel

Abstract

An in‐tandem study comprising two affinity chromatographic columns, Blue Avidgel P (for the selective removal of albumin) and an aza‐arenophilic gel (Gel A) containing a nitrogen capping nucleophile (for the selective removal of IgG), was conducted to obtain high purity IgG from serum. The results demonstrated that highly pure IgG (>97%) can be obtained from the phenethylamine capped aza‐arenophilic gel column after the serum was passed through the Blue Avidgel P column in contrast to the 38% IgG purity obtained when the serum was passed through a Gel A column alone under similar conditions. Further studies show that IgG selectivity is not dependent on the nature of the support material used in the synthesis of these aza‐arenophilic gels since the synthesis of aza‐arenophilic gels using either Sepharose or Fractogel yield similar results.     

Application of partial-filling capillary electrophoresis using lectins and glycosidases for the characterization of oligosaccharides in a therapeutic antibody

Oligosaccharides in therapeutic recombinant antibodies play important roles in regulation of various biological functions. To monitor the glycosylation profiles of antibody pharmaceuticals in the manufacturing process, a highly sensitive and specific method is required. We extended partial-filling techniques using lectins and exoglycosidases in capillary electrophoresis for the characterization of 8-aminopylene-1,3,6-trisulfonic acid labeled N-linked oligosaccharides derived from the therapeutic antibody rituximab. In the lectin-filling method, Galb1–4GlcNAc-specific Erythrina cristagali agglutinin, a1, 6-linked Fuc-specific Aleuria aurantia lectin and Neu5Aca2–3Gal-specific Maackia amurensis lectin were used. The oligosaccharides migrated through the lectin plug during separation; the changes in separation profiles were observed according to the interaction with the lectins. The glycosidase-filling method allowed rapid digestion as suggested by the electropherograms. Partial-filling CE methods can avoid tedious hands-on procedures such as overnight incubation and optimization reaction condition with lectins and exoglycosidases. Combination of these partial-filling capillary electrophoresis methods makes the characterization of oligosaccharide profiles of therapeutic antibodies easier and faster.     

Application of a pH responsive multimodal hydrophobic interaction chromatography medium for the analysis of glycosylated proteins

Protein glycosylation has significant effects on the structure and function of proteins. The efficient separation and enrichment of glycoproteins from complex biological samples is one key aspect and represents a major bottleneck of glycoproteome research. In this paper, we have explored pH multimodal hydrophobic interaction chromatography to separate glycosylated from non-glycosylated forms of proteins. Three different proteins, ribonuclease, invertase and IgG, have been examined and different glycoforms have been identified. The media itself shows strong responsiveness to small variations in pH, which makes it possible to fine-tune the chromatographic conditions according to the properties of the protein isolated. Optimal glycoprotein separation has been obtained at pH 4. The pH responsive multimodal HIC medium in contrast to conventional HIC media is able to resolve contaminating DNA.     

Preparation of thiophilic paramagnetic adsorbent for separation of antibodies

The micron-sized microspheres with superparamagnetic property were synthesized with vinyl acetate and divinylbenzene by microsuspension polymerization. After the complete alcoholysis, these hydroxyl-functionalized microspheres were activated by divinylfone and modified with mercaptoethanol to prepare the thiophilic magnetic adsorbent, which was used to specifically isolate immunoglobulin G （IgG） from human serum. This thiophilic magnetic adsorbent performed an evident salt-dependent adsorption behavior for IgG. Due to their salt-promoted adsorption towards IgG under high salt concentration, the absorbed antibodies could be extracted in low salt concentration with high purity.     

Studies on Human Immunoglobulin G from GBS Patient (Ⅲ)-The Determination of Molecular Weight of Human Immunoglobulin G by Capillary SDS Gel Electrophoresis

IntroductionGuillain-Barresyndrome(GBS)isconsideredtobeanautoimmunedisorderofperipheralnervoussystem'.Thebalanceofsympatheticnervesandparasympatheticnervesisdisturbed,whichleadstocompleteparalysisi'2'3.Moslpatientsshowthetypicalsymptomsafterbeinginfe...