Quality Of Recombinant Proteins Research Articles

Enhancing Recombinant Protein Quality and Yield by Protein Stability Profiling

The reliable production of large amounts of stable, high-quality proteins is a major challenge facing pharmaceutical protein biochemists, necessary for fulfilling demands from structural biology, for high-throughput screening, and for assay purposes throughout early discovery. One strategy for bypassing purification challenges in problematic systems is to engineer multiple forms of a particular protein to optimize expression, purification, and stability, often resulting in a nonphysiological sub-domain. An alternative strategy is to alter process conditions to maximize wild-type construct stability, based on a specific protein stability profile (PSP). ThermoFluor, a miniaturized 384-well thermal stability assay, has been implemented as a means of monitoring solution-dependent changes in protein stability, complementing the protein engineering and purification processes. A systematic analysis of pH, buffer or salt identity and concentration, biological metals, surfactants, and common excipients in terms of an effect on protein stability rapidly identifies conditions that might be used (or avoided) during protein production. Two PSPs are presented for the kinase catalytic domains of Akt-3 and cFMS, in which information derived from a ThermoFluor PSP led to an altered purification strategy, improving the yield and quality of the protein using the primary sequences of the catalytic domains.

Automated mass correction and data interpretation for protein open‐access liquid chromatography‐mass spectrometry

Characterization of recombinant protein purification fractions and final products by liquid chromatography-mass spectrometry (LC/MS) are requested more frequently each year. A protein open-access (OA) LC/MS system was developed in our laboratory to meet this demand. This paper compares the system that we originally implemented in our facilities in 2003 to the one now in use, and discusses, in more detail, recent enhancements that have improved its robustness, reliability, and data reporting capabilities. The system utilizes instruments equipped with reversed-phase chromatography and an orthogonal accelerated time-of-flight mass spectrometer fitted with an electrospray source. Sample analysis requests are accomplished using a simple form on a web-enabled laboratory information management system (LIMS). This distributed form is accessible from any intranet-connected company desktop computer. Automated data acquisition and processing are performed using a combination of in-house (OA-Self Service, OA-Monitor, and OA-Analysis Engine) and vendor-supplied programs (AutoLynx, and OpenLynx) located on acquisition computers and off-line processing workstations. Analysis results are then reported via the same web-based LIMS. Also presented are solutions to problems not addressed on commercially available, small-molecule OA-LC/MS systems. These include automated transforming of mass-to-charge (m/z) spectra to mass spectra and automated data interpretation that considers minor variants to the protein sequence-such as common post-translational modifications (PTMs). Currently, our protein OA-LC/MS platform runs on five LC/MS instruments located in three separate GlaxoSmithKline R&D sites in the US and UK. To date, more than 8000 protein OA-LC/MS samples have been analyzed. With these user friendly and highly automated OA systems in place, mass spectrometry plays a key role in assessing the quality of recombinant proteins, either produced at our facilities or bought from external sources, without dedicating extensive amounts of analyst resource.

The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures

Protein aggregation is a major bottleneck during the bacterial production of recombinant proteins. In general, the induction of gene expression at sub-optimal growth temperatures improves the solubility of aggregation-prone polypeptides and minimizes inclusion body (IB) formation. However, the effect of low temperatures on the quality of the recombinant protein, especially within the insoluble cell fraction, has been hardly ever explored. In this work, we have examined the conformational status of a recombinant GFP protein when produced in Escherichia coli below 37 degrees C. As expected, the fraction of aggregated protein largely decreased at lower temperatures, while the conformational quality of both soluble and aggregated GFP, as reflected by its specific fluorescence emission, progressively improved. This observation indicates that physicochemical conditions governing protein folding affect concurrently the quality of the soluble and the aggregated forms of a misfolding-prone protein, and that protein misfolding and aggregation are clearly not coincident events.

GlycoVis: Visualizing glycan distribution in the protein N‐glycosylation pathway in mammalian cells

Glycosylation has profound effects on the quality of recombinant proteins produced in mammalian cells. The biosynthetic pathways of N-linked glycans on glycoproteins involves a relatively small number of enzymes and nucleotide sugars. Many of these glycoconjugate enzymes can utilize multiple N-glycans as substrates, thus generating a large number of glycan intermediates, and making the biosynthetic pathway resemble a network with diverging and converging paths. The N-glycans on secreted glycoprotein molecules include not only terminal glycans, but also pathway intermediates. To better assess the glycan distribution and the potential route of their synthesis, we created GlycoVis, a visualization program that displays the distribution and the potential reaction paths leading to each N-glycan on the reaction network. The substrate specificities of the enzymes involved were organized into a relationship matrix. With the input of glycan distribution data, the program outputs a reaction pathway map which labels the relative abundance levels of different glycans with different colors. The program also traces all possible reaction paths leading to each glycan and identifies each pathway on the map. Glycoform distribution of Chinese Hamster Ovary cell-derived tissue plasminogen activator (TPA), and human and mouse IgG were used as illustrations for the application of GlycoVis. In addition, the intracellular and secreted IgG from an NS0 producer cell line were isolated, and their glycoform profiles were displayed using GlycoVis for comparison. This visualization tool facilitates the analysis of potential reaction paths utilized under different physiological or culture conditions, and may provide insight on the potential targets for metabolic engineering.

Quality of Recombinant Protein Determines the Amount of Autoreactivity Detected against the Tumor-Associated Epithelial Cell Adhesion Molecule Antigen: Low Frequency of Antibodies against the Natural Protein

The human epithelial cell adhesion molecule (EpCAM) is expressed on normal epithelial cells and is overexpressed in most carcinomas. EpCAM-targeted immunotherapy has been tried in several clinical studies. High titers of autoantibodies against EpCAM have been reported by different authors. We have generated large amounts of purified protein in S2 Drosophila cells (S2-EpCAM) with a purity of >96%. In contrast, the protein produced in baculovirus-dependent systems (baculo-EpCAM) that has been used in previous studies shows a purity of 79%. (1)H nuclear magnetic resonance spectrum of S2-EpCAM is typical of folded protein, whereas the baculo-EpCAM sample shows a spectrum corresponding to a partially unfolded protein. Using S2-EpCAM, denatured S2-EpCAM, and baculo-EpCAM, we measured EpCAM Abs of different isotypes in the serum of healthy controls and cancer patients. We found Ab titers against EpCAM in a much lower percentage of sera as published previously, and support the hypothesis that Ab reactivity in some published studies might be due to reactivity against denatured protein, to contaminating proteins in the baculovirus preparations, and to reactivity with BSA. Tetanus toxoid-reactive IgG Abs are present in 1000-fold higher titers compared with EpCAM-reactive Abs. Only IgA Abs were found in higher proportions and in higher concentrations than tetanus toxoid-specific Abs. Our study shows that EpCAM only rarely induces autoantibodies against native protein and emphasizes the importance of using extremely purified Ag preparations when evaluating Abs against tumor-associated Ags.

Enhancing therapeutic glycoprotein production in Chinese hamster ovary cells by metabolic engineering endogenous gene control with antisense DNA and gene targeting.

Recombinant glycoprotein therapeutics have proven to be invaluable pharmaceuticals for the treatment of chronic and life-threatening diseases. Although these molecules are extraordinarily efficacious, many diseases have high dosage requirements of several hundred milligrams of protein for each administration. Multiple doses at this level are often required for treatment. One of the major challenges currently facing the biotechnology industry is the development of large-scale, cost-effective production and manufacturing processes of these biologically synthesized molecules. Metabolic engineering of animal cell expression hosts promises to address this challenge by substantially enhancing recombinant protein quality, productivity, and biological activity. In this report, we describe a novel approach to metabolic engineering in Chinese hamster ovary cells by control of endogenous gene expression. Analysis of the advantages and limitations of using antisense DNA and gene targeting as a means of control are discussed and several gene candidates for regulation with these techniques are identified. Practical considerations for using these technologies to reduce the levels of the CHO cell sialidase (Warner et al., Glycobiology, 3, 455-463, 1993) as a model gene system for regulation are also presented.

A review of the biological significance of carbohydrates on glycoproteins and methods for their analysis.

Most secreted and cell surface proteins are glycosylated. There are three principal ways by which glycans may be attached to the protein: via the amino acid sequon of Asn-Xxx-Ser/Thr for N-glycosylation, the amino acids Ser/Thr for O-glycosylation and the C terminal amino acid in the GPI anchor of membrane bound glycoproteins. Typically a glycoprotein has greater than three glycan sites, each site containing between 10 and 30 structures. The presence of this many glycans on a single protein gives rise to the concept of glycoforms. Glycans on the protein can profoundly affect the 3-D structure of the protein and its biological function.1 The study of these glycans is therefore important whether one is involved in glycoprotein basic research, structure/function relationships, recombinant glycoprotein production or recombinant protein quality control.

Structural analysis of recombinant protein prepared by semi‐dry electroblotting after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis

Proteins that were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were electroblotted onto polyvinylidene difluoride membranes in procedures to prepare homogeneous recombinant proteins for direct N-terminal sequence analysis. A semi-dry blotting procedure was employed to immobilize protein bands on the membranes for subsequent sequence analysis. This method has been used routinely to evaluate the quality of recombinant proteins, which are present in crude cell extracts produced by different expression systems or under different expression conditions. N-Terminal processing, amino acid misincorporation, as well as the inefficient secretion of recombinant proteins can be detected by direct N-terminal sequence analysis of the purified electroblotted samples. Consequently, time-consuming chromatographic procedures can be eliminated. These procedures are also especially valuable for determining degradation sites of a purified recombinant protein, as well as evaluating multiple gene products expressed by isolated cluster genes.