Collaborative study for the establishment of Human immunoglobulin (molecular size) BRP replacement batches 4, 5 and 6.

Human immunoglobulin products are used for the treatment of a number of diseases, such as primary or secondary immunodeficiencies and autoimmune conditions due to the complete absence of antibodies or the production of defective immunoglobulins. Quality control of human immunoglobulin products is essential to ensure therapeutic functionality and safety. This includes testing for Fc function and anticomplementary activity (ACA), as well as verification of appropriate molecular size distribution using size-exclusion chromatography as prescribed in the European Pharmacopoeia (Ph. Eur.) monographs 0338, 0918, 2788 and 1928. To this end, specific biological reference preparations (BRPs) must be used. Stocks of the Ph. Eur. Human immunoglobulin (molecular size) BRP were running low and therefore a collaborative study was run by the European Directorate for the Quality of Medicines & HealthCare (EDQM), under the aegis of the Biological Standardisation Programme, to calibrate replacement batches. Eighteen laboratories, including manufacturers and Official Medicines Control Laboratories, took part in the study. Three batches of candidate BRPs were assessed and compared to Ph. Eur. Human immunoglobulin (molecular size) BRP 3 to ensure continuity. Based on the study results, the candidate BRPs were adopted by the Ph. Eur. Commission as Ph. Eur. Human immunoglobulin (molecular size) BRP batch 4, 5 and 6.

Semiquantitation of Paralytic Shellfish Toxins by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry Using Relative Molar Response Factors.

Paralytic shellfish toxins (PSTs) are a complex class of analogs of the potent neurotoxin saxitoxin (STX). Since calibration standards are not available for many PSTs, including C-11 hydroxyl analogs called M-toxins, accurate quantitation by liquid chromatography-mass spectrometry (LC-MS) can be challenging. In the absence of standards, PSTs are often semiquantitated using standards of a different analog (e.g., STX), an approach with a high degree of uncertainty due to the highly variable sensitivity between analytes in electrospray ionization. Here, relative molar response factors (RMRs) were investigated for a broad range of PSTs using common LC-MS approaches in order to improve the quantitation of PSTs for which standards are unavailable. First, several M-toxins (M1-M6, M9 and dcM6) were semipurified from shellfish using preparative gel filtration chromatography and quantitated using LC-charged aerosol detection (LC-CAD). The RMRs of PST certified reference materials (CRMs) and M-toxins were then determined using selective reaction monitoring LC-MS/MS and full scan LC-high-resolution MS (LC-HRMS) methods in positive and negative electrospray ionization. In general, RMRs for PSTs with similar chemical structures were comparable, but varied significantly between subclasses, with M-toxins showing the lowest sensitivity. For example, STX showed a greater than 50-fold higher RMR than M4 and M6 by LC-HRMS. The MS instrument, scan mode and polarity also had significant impacts on RMRs and should be carefully considered when semiquantitating PSTs by LC-MS. As a demonstration of their utility, the RMRs determined were applied to the semiquantitation of PSTs in contaminated mussels, showing good agreement with results from calibration with CRMs.

Morphology-transport relationships in liquid chromatography: Application to method development in size exclusion chromatography.

We present relationships between the multiscale structure and the separation properties of size exclusion chromatography (SEC) columns. Physical bed reconstructions of wall and bulk regions from a 2.1 mm i.d. column packed with fully porous 1.7 µm bridged-ethyl hybrid (BEH) particles, obtained by focused ion-beam scanning electron microscopy, serve as geometrical models for the packing microstructure in wall and central regions of a typical narrow-bore SEC column. In addition, the intraparticle mesopore space morphology of the BEH particles is reconstructed using electron tomography, to ultimately construct a realistic multiscale model of the bed morphology from mesopore level via interparticle macropore space to transcolumn scale. Complemented by the results of eddy dispersion simulations in computer-generated bulk packings, relationships between packing microstructure and transchannel, short-range interchannel, as well as transcolumn eddy dispersion are used to analyze the fluid dynamics in the interparticle macropore space of the model. Further, we simulate hindered diffusion and accessible porosity for passive, finite-size tracers in the intraparticle mesopore space, to finally determine the effective particle and bed diffusion coefficients of these tracers in the hierarchical (macro-mesoporous) bed. Retention and transport properties of polystyrene standards with hydrodynamic diameters from 5 to 95 Å in tetrahydrofuran are subsequently predicted without introducing bias from arbitrary models. These properties include the elution volumes of the polystyrene standards, the global peak capacity (over the entire separation window), and the rate of peak capacity at any fixed elution volume. Optimal flow rates yielding maximal global peak capacity and a nearly uniform rate of peak capacity over the entire separation window are close to 0.04 and 0.20 mL/min, respectively. SEC column performance obtained for fully porous and superficially porous particles is compared by varying the core-to-particle diameter ratio ρ from 0 to 0.95. Because the separation window is narrowing more rapidly than the rate of peak capacity is growing with increasing ρ, core-shell particles always provide smaller global peak capacity; they still can be advantageous but only for simple sample mixtures. The presented morphology-performance approach holds great promise for method development in SEC.

A Streamlined Bioanalytical Approach to Select a Compatible Primary Container System Early in Drug Development: A Toolbox for the Biopharmaceutical Industry.

To ensure drug efficacy and patient safety, the importance of interaction between primary container and a biological drug product must not be ignored. The United States Food and Drug Administration guidance on development and manufacturing of combination products (e.g., the biologic and the primary container) encourages careful selection and stability testing of the drug and its performance in the marketed primary container. With various options available for primary container type (vials, prefilled syringes, and cartridges), material (e.g., glass or plastic), and lubricants/coatings, we designed a platform consisting of several bioanalytical methods that can simplify selection of a compatible primary container. We tested the stability of a commercially available monoclonal antibody (mAb) in 3 syringe types under 3 conditions: cold storage, high temperature, and agitation induced stress, respectively. Under each condition, dynamic fluid imaging was sensitive enough to differentiate mAb stability as measured by aggregate formation in different syringe systems, followed by size exclusion-high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis but only at high temperature. With this platform, we identified a primary container that provided higher mAb stability under cold storage as well as stress conditions. We recommend that such an approach should be applied early in drug development stage to identify a superior primary container system to maintain drug stability and quality.

Development of a stable chemically cross-linked erythropoietin dimer for use in the quality control of erythropoietin therapeutic products.

Erythropoietin (EPO) is a glycoprotein hormone which promotes red cell replenishment and is also a global biotherapeutic medicine widely used to treat anaemia resulting, for example, from chemotherapy. Requirements of the European Pharmacopoeia stipulate that the level of dimer must be quantified in clinical EPO products (with a limit of 2%). Quantification is hampered by the lack of reference preparations containing stable measurable levels of EPO dimer, but the reproducible generation of a stable dimerised EPO preparation is challenging. We describe here the development of a lyophilised, chemically cross-linked EPO preparation, which has good stability and may be used for calibration and system suitability assurance for the size exclusion chromatographic separation of EPO preparations. Graphical abstract.

Serum Exosome Isolation by Size-Exclusion Chromatography for the Discovery and Validation of Preeclampsia-Associated Biomarkers.

Exosomes are extracellular nanovesicles of complex and heterogeneous composition that are released in biofluids such as blood. The interest in the characterization of exosomal biochemistry has increased over the last few years as they convey cellular proteins, lipids, and RNA that might reflect the biological or pathological condition of the source cell. In particular, association of changes of exosome proteins with specific pathogenic processes arises as a promising method to identify disease biomarkers as for the pregnancy-related preeclampsia. However, the overlapping physicochemical and structural characteristics of different types of extracellular vesicles have hindered the consolidation of universally accepted and standardized purification or enrichment protocols. Thus, it has been recently demonstrated that the exosomal protein profile resulting from in-depth proteomics analyses is highly dependent on the preparation protocol used, which will determine the particle type specificity and the presence/absence of contaminating proteins.In this chapter, an isolation method of serum exosomes based on size-exclusion chromatography (SEC) using qEV columns (Izon) is described. We show that this method is fast and reliable, as the population of exosomes isolated is homogeneous in terms of size, morphology, and protein composition. This exosome enrichment method is compatible with downstream qualitative and quantitative proteomic analysis of the samples.

An overview of the uses of high performance size exclusion chromatography (HPSEC) in the characterization of natural organic matter (NOM) in potable water, and ion-exchange applications.

Natural organic matter (NOM) constitutes the terrestrial and aquatic sources of organic plant like material found in water bodies. As of recently, an ever-increasing amount of effort is being put towards developing better ways of unraveling the heterogeneous nature of NOM. This is important as NOM is responsible for a wide variety of both direct and indirect effects: ranging from aesthetic concerns related to taste and odor, to issues related to disinfection by-product formation and metal mobility. A better understanding of NOM can also provide a better appreciation for treatment design; lending a further understanding of potable water treatment impacts on specific fractions and constituents of NOM. The use of high performance size-exclusion chromatography has shown a growing promise in its various applications for NOM characterization, through the ability to partition ultraviolet absorbing moieties into ill-defined groups of humic acids, hydrolysates of humics, and low molecular weight acids. HPSEC also has the ability of simultaneously measuring absorbance in the UV-visible range (200-350 nm); further providing a spectroscopic fingerprint that is simply unavailable using surrogate measurements of NOM, such as total organic carbon (TOC), ultraviolet absorbance at 254 nm (UV254), excitation-emission matrices (EEM), and specific ultraviolet absorbance at 254 nm (SUVA254). This review mainly focuses on the use of HPSEC in the characterization of NOM in a potable water setting, with an additional focus on strong-base ion-exchangers specifically targeted for NOM constituents.

Molecular Weight Determination of Aloe Polysaccharides Using Size Exclusion Chromatography Coupled with Multi-Angle Laser Light Scattering and Refractive Index Detectors.

Background: Size exclusion chromatography (SEC)/refractive index (RI) were used to determine molecular weight (MW) and molecular weight distributions (MWD) of polysaccharides. In aloe product research and quality control, commercially available pullulan and dextran are most commonly employed as calibration standards. Significant difference in the MW and MWD were found in literature when different methods were used. Objectives: This study was to investigate the traditional methods and more recent technologies used to determine the MW and MWD of Aloe vera polysaccharides. Methods: In this study, multi-angle laser light scattering (MALS) detection was studied on three polysaccharides, 1, 2, and 3, that were isolated and purified from A. vera leaf. The chemical structures of 1-3 were characterized as 1, 4-ß-linked glucomannans by monosaccharide composition and glycosidic linkage analysis. Absolute MW and root-mean-square radius were determined by MALS on the isolated aloe polysaccharides. The conditions to obtain reliable results from MALS measurement were examined. Results: MALS analysis demonstrates that the 1, 4-ß-linked glucomannan adopt the conformation of random coils or hard spheres in the analytical environment of a 0.1 M NaCl solution. Non-size exclusion effects and interactions between polysaccharide molecules were also observed in some aloe polysaccharides in the current analysis. The weight-average MW obtained by MALS measurement for 1, 2, and 3 are 55, 129, and 962 kDa, respectively. Comparing the results with SEC/RI calibrated by pullulan and dextran standards, marked differences in the MWD are found. Both overestimated the MW of 1 and 2 by factors of 4.4 and 4.2, and 2.4 and 1.6, when using dextran and pullulan calibration, respectively. Using pullulan calibration underestimated the MW of 3 by a factor of 3.1, but a similar result was obtained from dextran calibration compared to MALS measurement. The two isolated aloe polysaccharides were employed to be broad calibration standards or to be combined with narrow polydispersity pullulan calibration standards. Several aloe samples were tested using the different calibration curves, and the determined MWs were compared with the results obtained by MALS measurement. Conclusions: The results clearly indicated that until true polysaccharide standards become available MW and MWD's will be simply relative to the standards employed and the technologies used.

Conversion of calibration curves for accurate estimation of molecular weight averages and distributions of polyether polyols by conventional size exclusion chromatography.

Accurate measurement of molecular weight averages (M¯n,M¯w,M¯z) and molecular weight distributions (MWD) of polyether polyols by conventional SEC (size exclusion chromatography) is not as straightforward as it would appear. Conventional calibration with polystyrene (PS) standards can only provide PS apparent molecular weights which do not provide accurate estimates of polyol molecular weights. Using polyethylene oxide/polyethylene glycol (PEO/PEG) for molecular weight calibration could improve the accuracy, but the retention behavior of PEO/PEG is not stable in THF-based (tetrahydrofuran) SEC systems. In this work, two approaches for calibration curve conversion with narrow PS and polyol molecular weight standards were developed. Equations to convert PS-apparent molecular weight to polyol-apparent molecular weight were developed using both a rigorous mathematical analysis and graphical plot regression method. The conversion equations obtained by the two approaches were in good agreement. Factors influencing the conversion equation were investigated. It was concluded that the separation conditions such as column batch and operating temperature did not have significant impact on the conversion coefficients and a universal conversion equation could be obtained. With this conversion equation, more accurate estimates of molecular weight averages and MWDs for polyether polyols can be achieved from conventional PS-THF SEC calibration. Moreover, no additional experimentation is required to convert historical PS equivalent data to reasonably accurate molecular weight results.

The NISTmAb Reference Material 8671 value assignment, homogeneity, and stability.

The NISTmAb Reference Material (RM) 8671 is intended to be an industry standard monoclonal antibody for pre-competitive harmonization of best practices and designing next generation characterization technologies for identity, quality, and stability testing. It must therefore embody the quality and characteristics of a typical biopharmaceutical product and be available long-term in a stable format with consistent product quality attributes. A stratified sampling and analysis plan using a series of qualified analytical and biophysical methods is described that assures RM 8671 meets these criteria. Results for the first three lots of RM 8671 highlight the consistency of material attributes with respect to size, charge, and identity. RM 8671 was verified to be homogeneous both within and between vialing lots, demonstrating the robustness of the lifecycle management plan. It was analyzed in concert with the in-house primary sample 8670 (PS 8670) to provide a historical link to this seminal material. RM 8671 was verified to be fit for its intended purpose as a technology innovation tool, external system suitability control, and cross-industry harmonization platform. Graphical abstract The NISTmAb Reference Material (RM) 8671 is intended to be an industry standard monoclonal antibody for pre-competitive harmonization of best practices and designing next generation characterization technologies for identity, quality, and stability testing.

Development of orthogonal NISTmAb size heterogeneity control methods.

The NISTmAb is a monoclonal antibody Reference Material from the National Institute of Standards and Technology; it is a class-representative IgG1κ intended to serve as a pre-competitive platform for harmonization and technology development in the biopharmaceutical industry. The publication series of which this paper is a part describes NIST's overall control strategy to ensure NISTmAb quality and availability over its lifecycle. In this paper, the development of a control strategy for monitoring NISTmAb size heterogeneity is described. Optimization and qualification of size heterogeneity measurement spanning a broad size range are described, including capillary electrophoresis-sodium dodecyl sulfate (CE-SDS), size exclusion chromatography (SEC), dynamic light scattering (DLS), and flow imaging analysis. This paper is intended to provide relevant details of NIST's size heterogeneity control strategy to facilitate implementation of the NISTmAb as a test molecule in the end user's laboratory. Graphical abstract Representative size exclusion chromatogram of the NIST monoclonal antibody (NISTmAb). The NISTmAb is a publicly available research tool intended to facilitate advancement of biopharmaceutical analytics. HMW = high molecular weight (trimer and dimer), LMW = low molecular weight (2 fragment peaks). Peak labeled buffer is void volume of the column from L-histidine background buffer.

Size exclusion chromatography of lignin: The mechanistic aspects and elimination of undesired secondary interactions.

Characterization of lignin and its degradation products, more specifically determination of their molecular weight (MW) distribution, is essential for assessment and applications of these potentially renewable phenolics. Several representative gel filtration and gel permeation systems were evaluated in this work focusing on understanding of undesired secondary non-SEC interactions while utilizing four sets of commercially available polymeric standards as well as low-MW lignin model compounds including diarene standards synthesized in-house. The gel permeation column with a nonpolar highly cross-linked porous polystyrene/divinylbenzene-based stationary phase provided the most effective separation by MW for both low and high MW model compounds. Notably, the column with a higher pore and lower particle size provided a better resolution towards polymeric standards, even though the particle size effect was downplayed in the earlier SEC studies of lignin. For two other evaluated gel filtration and gel permeation columns, the separation was strongly affected by functionalities of the analytes and correlated with the compounds' pKa rather than MW. We showed that the separation on the stationary phases featuring polar hydroxyl groups led to specific column-analyte secondary interactions, perhaps based on their hydrogen bonding with lignin. Further, the SEC column evaluation yielded similar results with two sets of chemically different standards. This setup may be used as a general approach to selecting an applicable column for lignin SEC analysis. We confirmed the obtained results with a different independent method implementing a novel approach for lignin number-average MW (Mn) calculation based on laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF-MS) data. The determined Mn corroborated the SEC results.

Stability assessment of antibody-drug conjugate Trastuzumab emtansine in comparison to parent monoclonal antibody using orthogonal testing protocol.

Antibody-drug conjugates (ADC) represent an emerging, novel class of biopharmaceuticals. The heterogeneity originating from the sophisticated structure requires orthogonal analytical techniques for quality and stability assessment of ADC to ensure safety and efficacy. In this study, the stability of Trastuzumab (recombinant humanized IgG1 mAb, targeting HER2 receptor) and its ADC with DM1 (anti-tubulin anticancer drug), Trastuzumab emtansine (T-DM1) were studied. SE-HPLC was used to monitor formation of aggregates and/or fragments of the monoclonal antibodies (mAb). Correlation with the results of reducing and non-reducing sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) and dynamic light scattering (DLS) were performed to interpret the obtained results. RP-HPLC was used for assessment of the stability of DM1 in ADC while spectrophotometry was employed to determine drug antibody ratio (DAR) . The studied drugs were subjected to several stress conditions including pH, temperature, mechanical agitation and repeated freeze and thaw to generate possible degradation products and ensure suitability of the assay protocol. The degradation pattern and extent were demonstrated under the indicated stress conditions. The correlation between the results of SE-HPLC and those of SDS-PAGE and DLS ensured the validity of the orthogonal assay protocol and indicated aggregates that were not detected using SE-HPLC. Results showed clearly that T-DM1 is relatively less stable than its parent mAb. This was attributed to the presence of the drug-linker part that is attached to the mAb. RP-HPLC showed that the cytotoxic drug moiety is liable for degradation under the studied conditions resulting in alteration of DAR as well as formation of degradation products. This confirmed the need for more robust coupling chemistries for production of safe and effective ADC and highlighted the importance of orthogonal testing protocols for quality assessment. The assay protocol should be applicable for quality and stability assessment of various ADC.

Improving the accuracy of hyaluronic acid molecular weight estimation by conventional size exclusion chromatography.

There is an unreasonably high variation in the literature reports on molecular weight of hyaluronic acid (HA) estimated using conventional size exclusion chromatography (SEC). This variation is most likely due to errors in estimation. Working with commercially available HA molecular weight standards, this work examines the extent of error in molecular weight estimation due to two factors: use of non-HA based calibration and concentration of sample injected into the SEC column. We develop a multivariate regression correlation to correct for concentration effect. Our analysis showed that, SEC calibration based on non-HA standards like polyethylene oxide and pullulan led to approximately 2 and 10 times overestimation, respectively, when compared to HA-based calibration. Further, we found that injected sample concentration has an effect on molecular weight estimation. Even at 1g/l injected sample concentration, HA molecular weight standards of 0.7 and 1.64MDa showed appreciable underestimation of 11-24%. The multivariate correlation developed was found to reduce error in estimations at 1g/l to <4%. The correlation was also successfully applied to accurately estimate the molecular weight of HA produced by a recombinant Lactococcus lactis fermentation.

Evaluation of automated Wes system as an analytical and characterization tool to support monoclonal antibody drug product development.

Monitoring and evaluation of critical quality attributes (cQA) in monoclonal antibodies (mAb) are a regulatory requirement in pharmaceutical industry. High molecular weight (HMW) species are of critical importance due to the potential risk associated with immunogenicity. HMW species are typically monitored by size exclusion chromatography (SEC). Although low molecular weight (LMW) species are also detected by SEC, low-resolution separation of LMW limits its capability to monitor mAb fragmentation. Recently, we have developed new methods for LMW characterization and evaluation based on the Wes instrument from ProteinSimple. The capillary western blot is based upon size-based separation in a capillary system, and detection by specific immunoprobing, following the separation. The capability of this method for characterization of mAb fragments were demonstrated. The characterization was achieved by probing two antibodies targeted to specific regions (Fc region or Fab region) of IgG1 protein. The specificity of these two antibodies was evaluated against F (ab') 2 and Fc/2 fragments generated from Ides enzyme treated IgG1 protein. The results showed the selected antibodies provide high specificity to F (ab') 2 and Fc/2 fragments. Fractions collected from SEC were used to evaluate this method. The detected fragments from SEC fractions were identified based on their estimated molecular weight and antibody detection. The result proved the capability of the capillary western blot as a characterization method for IgG1 fragments. In addition, with the specific detection to IgG1 and IgG4, the power of the capillary western blot to specifically characterize and evaluate individual IgG fragmentations in an IgG1 and IgG4 mixture was also demonstrated. When heat stressed samples were used, results showed method capability as stability indicating in IgG1 and IgG4 mixture samples. The stressed mixture samples were also evaluated by the total protein assay in which protein samples were biotinylated after separation and were labeled with HRP linked streptavidin to provide chemiluminescence detection. The results indicated total protein assay can be a useful complementary method to capillary western blot immunoassay.

Evaluation of size exclusion chromatography columns packed with sub-3µm particles for the analysis of biopharmaceutical proteins.

The aim of this study was to evaluate the practical possibilities and limitations of several recently introduced size exclusion chromatographic (SEC) columns of 150×4.6mm, sub-3µm (Agilent AdvanceBioSEC 2.7µm, Tosoh TSKgel UP-SW3000 2.0µm, Phenomenex Yarra SEC X-150 1.8µm and Waters Acquity BEH200 1.7µm) for the separation of biopharmaceutical proteins. For this purpose, some model proteins were tested, as well as several commercial therapeutic monoclonal antibodies (mAbs) and antibody-drug-conjugates (ADCs). Calibration curves were drawn to highlight the applicability of these new SEC columns for the separation of mAbs, ADCs and their aggregates, despite some differences in their nominal pore diameter (vary from 150 to 300Å). The kinetic performance (van Deemter curves and kinetic pots) was evaluated. Columns packed with 1.7-2.0µm particles improved the plate count by a factor of 1.5-2 compared to 2.7µm particles, which is in agreement with theoretical expectations. Finally, possible secondary hydrophobic and/or electrostatic interactions between the SEC stationary phases and biopharmaceutical proteins were systematically studied. Significant differences in nonspecific interactions were observed, with hydrophobic interactions generally exerting more influence than electrostatic interactions. The use of a novel bond chemistry with the AdvanceBioSEC column was found highly effective to limit non-specific interactions and pave the way to further improvements for column provider. At the end, the average resolutions achieved on the four sub-3µm SEC columns between monomer and dimer structures were comparable for ten approved mAbs products.

The importance of system band broadening in modern size exclusion chromatography.

In the last few years, highly efficient UHP-SEC columns packed with sub-3µm particles were commercialized by several providers. Besides the particle size reduction, the dimensions of modern SEC stationary phases (150×4.6mm) was also modified compared to regular SEC columns (300×6 or 300×8mm). Because the analytes are excluded from the pores in SEC, the retention factors are very low, ranging from -1

Filling the gap: Calibration of the low molar-mass range of cellulose in size exclusion chromatography with cello-oligomers.

Degraded celluloses are becoming increasingly important as part of product streams coming from various biorefinery scenarios. Analysis of the molar mass distribution of such fractions is a challenge, since neither established methods for mono- or disaccharides nor common methods for polysaccharide characterization cover the intermediate oligomer range appropriately. Size exclusion chromatography (SEC) with multi-angle laser light scattering (MALLS), the standard approach for celluloses, suffers from decreased scattering intensities in the lower-molar mass range. The limitation in the low-molecular range can, in principle, be overcome by calibration, but calibration standards for such "short" celluloses are either not readily available or structurally remote and thus questionable. In this paper, we present the calibration of a SEC system- for the first time - with monodisperse cellooligomer standards up to about 3400gmol-1. These cellooligomers are "short-chain celluloses" and can be seen as the "true" standard compounds, by contrast to commonly used standards that are chemically different from cellulose, such as pullulan, dextran, polystyrene, or poly(methyl methacrylate). The calibration is compared against those commercial standards and correction factors are calculated. Calibrations with non-cellulose standards can now be adjusted to yield better fitting results, and data already available can be corrected retrospectively.

Interpretation of size-exclusion chromatography for the determination of molecular-size distribution of human immunoglobulins.

Molecular-size distribution by size-exclusion chromatography (SEC) [1] is used for the quantification of unwanted aggregated forms in therapeutic polyclonal antibodies, referred to as human immunoglobulins (Ig) in the European Pharmacopoeia. Considering not only the requirements of the monographs for human normal Ig (0338, 0918 and 2788) [2-4], but also the general chapter on chromatographic techniques (2.2.46) [5], several chromatographic column types are allowed for performing this test. Although the EDQM knowledge database gives only 2 examples of suitable columns as a guide for the user, these monographs permit the use of columns with different lengths and diameters, and do not prescribe either particle size or pore size, which are considered key characteristics of SEC columns. Therefore, the columns used may differ significantly from each other with regard to peak resolution, potentially resulting in ambiguous peak identity assignment. In some cases, this may even lead to situations where the manufacturer and the Official Medicines Control Laboratory (OMCL) in charge of Official Control Authority Batch Release (OCABR) have differing molecular-size distribution profiles for aggregates of the same batch of Ig, even though both laboratories follow the requirements of the relevant monograph. In the present study, several formally acceptable columns and the peak integration results obtained therewith were compared. A standard size-exclusion column with a length of 60 cm and a particle size of 10 µm typically detects only 3 Ig fractions, namely monomers, dimers and polymers. This column type was among the first reliable HPLC columns on the market for this test and very rapidly became the standard for many pharmaceutical manufacturers and OMCLs for batch release testing. Consequently, the distribution of monomers, dimers and polymers was established as the basis for the interpretation of the results of the molecular-size distribution test in the relevant monographs. However, modern columns with a smaller particle size provide better resolution and also reveal a class of components designated here as oligomers. This publication addresses the interpretation of the SEC test for Ig with respect to the following questions: - how can molecular-size distribution tests benefit from the use of the most recent column technology without changing the sense of well-established quality parameters? - is it possible to mathematically define a way to interpret chromatograms generated with various column types with the same fractionation range but different resolution power? - how should oligomers be considered regarding compliance with compendial specifications?

Macroprolactinaemia: a biological diagnostic strategy from the study of 222 patients.

OBJECTIVES: Gel filtration chromatography (GFC), the gold standard for macroprolactinaemia (MPRL) diagnosis, is a slow, costly and labour-intensive method. To limit the number of GFC required, we evaluated two screening tests for MPRL: prolactin (PRL) recovery after polyethylene glycol (PEG) precipitation and PRL concentration ratio, derived from two assays, each having different big-big-PRL cross-reactivities.In some patients, MPRL is characterised by clinical symptoms which can be associated with an excess of monomeric PRL. We compared the monomeric PRL concentration obtained from GFC with the PRL concentration i) on a cobas e 601 analyser and ii) in the supernatant after PEG precipitation. DESIGN AND METHODS: We studied hyperprolactinaemic sera subjected to physician-ordered GFC, between February 2013 and July 2014. We performed PEG precipitation (to evaluate the PRL concentration and rate of recovery in the supernatant) and two PRL assays: RIA and electrochemiluminescent assay (ECLIA), on a Roche cobas e 601 analyser, and calculated the RIA/ECLIA ratio. RESULTS: Among the 222 sera, we were able to diagnose or exclude MPRL in 72.1% of cases, based solely on the ratio and/or recovery. In the remaining cases, GFC was necessary for making a diagnosis. Elevated monomeric PRL was present in 10.9% of macroprolactinaemic sera. In the case of MPRL, both PRL measurements on the cobas analyser and in the supernatant weakly correlated with monomeric PRL values obtained from GFC. CONCLUSIONS: The combination of PEG and RIA/ECLIA ratio analysis reduced the number of necessary GFC. However, GFC is essential in MPRL cases to evaluate the monomeric PRL concentration.