Statistical properties of the band-broadening function.

Statistical properties of the band-broadening function calculated from the equilibrium and kinetic theories of Giddings and Eyring are compared with real liquid-chromatography elution curves of low-molecular-weight substances and with light-scattering elution curves of a high-molecular-weight polystyrene standard injected at several concentrations. The curves obtained by liquid chromatography agree in a large span of elution times with the theoretical ones and can be described by one value of the plate-height. The size exclusion chromatography (SEC) elution curves seem to approach the predicted statistical properties with decreasing concentration of injected polymer but, in general, a reliable prediction of band-broadening (BB) functions in SEC of polymers, especially of non-symmetric BB functions near the exclusion limit, seems not possible at this point. The relation of the theory with the characteristic-function approach developed by Dondi is also discussed.

Simulation of size-exclusion chromatography distribution coefficients of comb-shaped molecules in spherical pores comparison of simulation and experiment.

Simulations of the distribution coefficients of linear polymers and regular combs with various spacings between the arms have been performed. The distribution coefficients were plotted as a function of the number of segments in order to compare the size exclusion chromatography (SEC)-elution behavior of combs relative to linear molecules. By comparing the simulated SEC-calibration curves it is possible to predict the elution behavior of comb-shaped polymers relative to linear ones. In order to compare the results obtained by computer simulations with experimental data, a variety of comb-shaped polymers varying in side chain length, spacing between the side chains and molecular weights of the backbone were analyzed by SEC with light-scattering detection. It was found that the computer simulations could predict the molecular weights of linear molecules having the same retention volume with an accuracy of about 10%, i.e. the error in the molecular weight obtained by calculating the molecular weight of the comb-polymer based on a calibration curve constructed using linear standards and the results of the computer simulations are of the same magnitude as the experimental error of absolute molecular weight determination.

Determination of the size distribution of liposomes by SEC fractionation, and PCS analysis and enzymatic assay of lipid content.

In this study, small liposomes obtained by high-pressure homogenization were fractionated according to their particle sizes by size exclusion chromatography (SEC). The subfractions were analyzed by photon correlation spectroscopy (PCS) as well as enzymatic phosphatidylcholine (PC) assay for their particle sizes and lipid contents, respectively. For small egg PC-liposomes, a size range of 15 nm to 60 nm was found, with 80% of the vesicles being smaller than 30 nm in size. This is in contradiction to a mean size of 85 +/- 32 nm as indicated by PCS without fractionation. The PCS technique appears to underestimate very small particles below 30 nm if (few) bigger particles are present. The PCS particle size analysis of unfractionated hydrogenated egg PC/cholesterol-liposomes (2:1, mole/mole) by PCS did not yield any significant results. On fractionation, however, a particle size range of 40 nm to 120 nm was determined in a reproducible manner. Our results indicate that the combination of size exclusion fractionation with subsequent photon correlation spectroscopic particle size analysis and enzymatic PC assay can give both more detailed and more reliable insight into the particle size distribution of small liposomes than PCS alone.

Process characterization for metal-affinity chromatography of an Fc fusion protein: a design-of-experiments approach.

The utility of a design-of-experiments approach was investigated for process characterization of a metal-affinity chromatographic purification process for an Fc fusion protein. This approach gave a better understanding of some of the key process variables as well as robustness for this step in the purification process. Single-variable experiments were employed to screen some of the potentially important variables in this step. Ranges for these variables were set based on prior experience in clinical manufacturing with similar processes. Following these experiments, a fractional factorial study was employed to further investigate the most important variables and their interactions. Key operational variables that had an impact on step yield and eluate purity were identified. In addition, the study helped identify a worst-case scenario for the step purity and helped assure that the rest of the process would successfully purify the product. This paper demonstrates the utility of a design-of-experiments approach for the characterization and validation of process chromatography steps in downstream processing. In addition, this study emphasizes the utility of robustness studies early in process development and establishes a strategy for future robustness studies.

[An immunoglobulin preparation as a calibrating agent for the gel filtration method]. / Preparat immunoglobulina v kachestve kalibranta dlia metoda gel-fil'tratsii.

The fractional composition of commercial immunoglobulin was studied by the method of gel filtration on ultragel AcA-34 and the possibility of its use for the calibration of a chromatographic column was shown. The fractionation of a specimen of IgG revealed the presence of 4 fractions. Their molecular weights corresponded to dimers, monomers, Fab fragments and low-molecular peptides. The qualitative fractional composition of the preparation was shown to be stable during 3 years of storage, as well as after keeping the preparation at 37 degrees C for 1 month. The attested characteristic of each IgG fraction, the distribution coefficient (Kav), was established. The use of a specimen of immunoglobulin obtained with the use of the modified Cohn's method--as calibrant will make it possible to calibrate the columns for a shorter period and to control the correctness of the analysis.

A routine high-performance size-exclusion chromatography to determine molecular size distribution of Haemophilus influenzae type b conjugate vaccines.

High-performance size exclusion chromatography has been used to determine the molecular size distribution of Haemophilus influenzae type b (Hib) conjugate vaccines. Both high molecular weight preparations of native Hib capsular polysaccharide coupled to tetanus toxoid and low molecular weight vaccines with Hib oligosaccharides linked to the CRM197 nontoxic mutant diphtheria protein were analysed. Columns with different fractionation ranges were used for the two kinds of vaccines. This method showed to be rapid, accurate and reproducible for different lots of Hib vaccine of different composition produced by various manufacturers. It could replace more time-consuming chromatographic methods enabling control authorities to employ a single methodological approach for different Hib vaccines.

[The molecular parameters of a meningococcal serogroup B protein-polysaccharide vaccine]. / Izuchenie molekuliarnykh parametrov meningokokkovoi belkovo-polisakharidnoi vaktsiny serogruppy B.

Methods for the evaluation of the molecular parameters of B polysaccharide (B-PS) in meningococcal protein-polysaccharide vaccine of group B are proposed. The comparison of two proposed methods, the passive hemagglutination inhibition test and rocket immunoelectrophoresis (RIEP), has shown that the latter method has the highest degree of correlation with the chemical method of the detection of B-PS, which is often hindered by lactose added as a bulking agent. RIEP may be recommended for the standardization and control of the commercial preparations of group B meningococcal vaccine. B-PS contained in the vaccine is known to be in a highly polymeric state: its yield is 66.4 +/- 1.7% to Kd = 0.25 and 85.3 +/- 1.2% to Kd = 0.5. B-PS contained in the prepared vaccine has been found to be highly stable. Outer membrane proteins of meningococci, forming a noncovalent complex with capsular B-PS, seem to stabilize its structure and prevent the depolymerization of molecules.

Determination of polymerized triglycerides in frying fats and oils by gel permeation chromatography: interlaboratory study.

An interlaboratory study of the gel permeation chromatographic (GPC) determination of polymerized triglycerides (PTG) in frying fats and oils was conducted by the Project Group on Collaborative Studies (PCS) of the Inspectorate for Health Protection, Food Inspection Service, The Netherlands. Thirteen laboratories participated in this study, which was focused on thermally abused oil and fat samples containing dimerized and polymerized triglycerides at levels just below and far above the Dutch regulatory limit of 16% (m/m). Samples were dissolved in tetrahydrofuran (THF) and analyzed on a GPC system, using THF as the mobile phase. Refractive index (RI) detection was used to determine PTG. Six heat-processed fat samples containing from 14 to 28% (m/m) PTG at 3 different levels (3 pairs of split level samples) were single analyzed according to the proposed procedure. Data analysis was performed according to the International Union of Pure and Applied Chemistry/International Organization for Standardization/AOAC protocols for statistics. The results of 2 collaborators were identified as outliers at the 20% (m/m) PTG level by applying the paired Grubbs test at the 1% level of significance. The repeatability relative standard deviation (RSDr) values ranged from 0.48 to 2.25%, whereas the reproducibility relative standard deviation (RSDR) values ranged from 1.34 to 4.57%.

Gel-test: interpretation and value of a new technique for the detection of irregular antibodies.

Here we report on our experience with the use of a 'Gel-Test' (DiaMed-ID Micro Typing System) technique for the detection and identification of irregular antibodies in a general hospital. This easy-to-use, standardized technique poses the question of the impact of its sensitivity on the specificity of the results. Of the 10% of observed positive reactions, 3.7% were irregular antibodies, 3.8% papain auto-antibodies, 1% cold antibodies and 2% not elucidated. Two hundred and eighteen irregular antibodies identified and titred with the 'gel-test' system were tested in parallel by 'tube' method. Sixty-three of these antibodies (29%) were not detected by the 'tube' method. While anti-Kell was always detected by both methods, we found the following false natives with the tube method: 15% anti-D, 32% anti-E, 42% anti-Cw and 58% anti-Lea. 68% of these false negatives had a low titre. The immunoglobulin class of the anti-E was studied; the sensitivity of the 'gel-test' system was associated with IgM in the anti-E. The sensitivity and standardization of the 'gel-test' technique guarantee greater safety in blood transfusion and increase efficiency in the prevention of foeto-maternal stimulation of anti-D.