Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications-Updated and completely revised edition.

This review is in support of the development of selective, precise, fast, and validated capillary electrophoresis (CE) methods. It follows up a similar article from 1998, Wätzig H, Degenhardt M, Kunkel A. "Strategies for capillary electrophoresis: method development and validation for pharmaceutical and biological applications," pointing out which fundamentals are still valid and at the same time showing the enormous achievements in the last 25 years. The structures of both reviews are widely similar, in order to facilitate their simultaneous use. Focusing on pharmaceutical and biological applications, the successful use of CE is now demonstrated by more than 600 carefully selected references. Many of those are recent reviews; therefore, a significant overview about the field is provided. There are extra sections about sample pretreatment related to CE and microchip CE, and a completely revised section about method development for protein analytes and biomolecules in general. The general strategies for method development are summed up with regard to selectivity, efficiency, precision, analysis time, limit of detection, sample pretreatment requirements, and validation.

An interlaboratory capillary zone electrophoresis-UV study of various monoclonal antibodies, instruments, and ε-aminocaproic acid lots.

Capillary zone electrophoresis ultraviolet (CZE-UV) has become increasingly popular for the charge heterogeneity determination of mAbs and vaccines. The ε-aminocaproic acid (eACA) CZE-UV method has been used as a rapid platform method. However, in the last years, several issues have been observed, for example, loss in electrophoretic resolution or baseline drifts. Evaluating the role of eACA on the reported issues, various laboratories were requested to provide their routinely used eACA CZE-UV methods, and background electrolyte compositions. Although every laboratory claimed to use the He et al. eACA CZE-UV method, most methods actually deviate from He's. Subsequently, a detailed interlaboratory study was designed wherein two commercially available mAbs (Waters' Mass Check Standard mAb [pI 7] and NISTmAb [pI 9]) were provided to each laboratory, along with two detailed eACA CZE-UV protocols for a short-end, high-speed, and a long-end, high-resolution method. Ten laboratories participated each using their own instruments, and commodities, showing excellence method performance (relative standard deviations [RSDs] of percent time-corrected main peak areas from 0.2% to 1.9%, and RSDs of migration times from 0.7% to 1.8% [n = 50 per laboratory], analysis times in some cases as short as 2.5 min). This study clarified that eACA is not the main reason for the abovementioned variations.

Combination of strong anion exchange liquid chromatography with microchip capillary electrophoresis sodium dodecyl sulfate for rapid two-dimensional separations of complex protein mixtures.

Two-dimensional separations provide a simple way to increase the resolution and peak capacity of complex protein separations. The feasibility of a recently developed instrumental approach for two-dimensional separations of proteins was evaluated. The approach is based on the general principle of two-dimensional gel electrophoresis. In the first dimension, semi-preparative strong anion exchange high-performance liquid chromatography is utilized and fractions are collected by means of a fraction collector. They are subsequently analyzed in the second dimension with microchip capillary electrophoresis sodium dodecyl sulfate. Microchip capillary electrophoresis provides the necessary speed (approximately 1 min/fraction) for short analysis. In this study, three different samples were investigated. Different constructs of soluble guanylyl cyclase were expressed in Sf9-cells using the baculovirus expression system. Cell lysates were analyzed and the resulting separations were compared. In our experimental setup, the soluble guanylyl cyclase was identified among hundreds of other proteins in these cell lysates, indicating its potential for screening, process control, or analysis. The results were validated by immunoblotting. Samples from Chinese hamster ovary cell culture before and after a purification step were investigated and approximately 9% less impurities could be observed. The separation patterns obtained for human plasma are closely similar to patterns obtained with two-dimensional gel electrophoresis and a total of 218 peaks could be observed. Overall, the approach was well applicable to all samples and, based on these results, further directions for improvements were identified. .

Protein analysis and stability: Overcoming trial-and-error by grouping according to physicochemical properties.

Today proteins are possibly the most important class of substances. Yet new tasks for proteins are still often solved by trial-and-error approaches. However, in some areas these euphemistically called "screening approaches" are not suitable. E.g. stability tests just take too long and therefore require a more strategic, target-orientated concept. This concept is available by grouping proteins according to their physicochemical properties and then pulling out the right drawer for new tasks. These properties include size, then charge and hydrophobicity as well as their patchinesses, and the degree of order. In addition, solubility, the content of (free) enthalpy, aromatic-amino-acid- and α/ß-frequency as well as helix capping, and corresponding patchiness, the number of specific motifs and domains as well as the typical concentration range can be helpful to discriminate between different groups of proteins. Analyzing correlations will reduce the necessary amount of parameters and additional ones, which may be still undiscovered at the present time, can be identified looking at protein subgroups with similar physicochemical properties which still behave heterogeneously. Step-by-step the methodology will be improved. Possibly protein stability will be the driver of this process, but all other areas such as production, purification and analytics including sample pre-treatment and the choice of appropriate separation conditions for e.g. chromatography and electrophoresis will profit from a rational strategy.

Bone Morphogenetic Protein 2 (BMP-2) Aggregates Can be Solubilized by Albumin-Investigation of BMP-2 Aggregation by Light Scattering and Electrophoresis.

Bone morphogenetic protein 2 (BMP-2) has a high tendency to aggregate at physiological pH and physiological ionic strength, which can complicate the development of growth factor delivery systems. The aggregation behavior in differently concentrated BMP-2 solutions was investigated using dynamic and static light scattering. It was found that at higher concentrations larger aggregates are formed, whose size decreases again with increasing dilution. A solubilizing effect and therefore less aggregation was observed upon the addition of albumin. Imaged capillary isoelectric focusing and the simulation of the surface charges of BMP-2 were used to find a possible explanation for the unusually low solubility of BMP-2 at physiological pH. In addition to hydrophobic interactions, attractive electrostatic interactions might be decisive in the aggregation of BMP-2 due to the particular distribution of surface charges. These results help to better understand the solubility behavior of BMP-2 and thus support future pharmaceutical research and the development of new strategies for the augmentation of bone healing.

A systematic and methodical approach for the efficient purification of recombinant protein from silkworm larval hemolymph.

The silkworm, Bombyx mori, is a promising expression system for the production of recombinant proteins, but the purification of these proteins is not easy because of the large amount of host proteins present. To investigate purity, recovery and scale-up ability of the purification of recombinant proteins expressed in silkworm larval hemolymph without any affinity tags, we used mCherry, a red fluorescence protein, as a model. The host cell proteins could be greatly reduced using a three-step chromatography protocol consisting of hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC) and heparin chromatography after heat pretreatment. The thermal treatment had the greatest impact on the removal of host cell extracellular proteins and increasing purity. There were still some minor traces of host cell proteins in the purified sample, which showed that the purification of recombinant proteins from the silkworm hemolymph was still challenging. The proposed protocol and affinity tag purification reduced the overall protein content by 99.84% and 99.95%, respectively, while the amount of DNA was reduced by 98.41% and 99.53%, respectively. Purities of our proposed protocol based on SDS-PAGE and capillary electrophoresis (CE) analyses were 85.45% and 43.60%, respectively, while those of Strep-tag affinity purification were 100% or 63.69%, respectively. Using densitometry, the overall recovery was calculated was 5.78%, which was higher than 4.09% using Strep-tag affinity purification. This proposed protocol, mainly based on thermal treatment, HIC, SEC and HiTrap Heparin HP column chromatography, is applicable to an upscalable purification for the silkworm expression system without employing affinity tag chromatography process.

Comparative charge-based separation study with various capillary electrophoresis (CE) modes and cation exchange chromatography (CEX) for the analysis of monoclonal antibodies.

Charge heterogeneity is an important critical quality attribute for the analysis of monoclonal antibodies (mAbs). For this, (imaged) capillary isoelectric focusing ((i)cIEF), ion exchange chromatography (IEC) and, recently, capillary zone electrophoresis (CZE) are the predominantly used techniques. In order to investigate which one is most suitable to answer a specific analytical question, here, the four aforementioned separation techniques were systematically evaluated using NISTmAb and Infliximab as test molecules. The performance parameters (precision, separation efficiency, linearity and sensitivity) were determined under comparable conditions. Moreover, important aspects for daily routine such as speed and ease of use were considered. Each technique has its own pros and cons. The (i)cIEF methodology is distinguished by its excellent separation efficiency. In addition, the native fluorescence mode in icIEF is a good tool to analyze small sample amounts (LOQ: 2.8 mg/l for Infliximab). Nevertheless, high performance liquid chromatography (HPLC) still has superior precision. CZE, and also micellar electrokinetic chromatography (MEKC), have emerged as further interesting alternatives. For all techniques, variations connected to the sample preparation strongly influence precision. Looking at the relative standard deviation (RSD) values of the relative peak areas, all techniques provide acceptable performance (RSD: 0.6-1.6%).