Large-scale Chromatography Research Articles

Scale-Up of Protein Purification: Downstream Processing Issues.

Large-scale chromatography operations continue to occupy the central position in the overall strategy for downstream processing and purification of therapeutic protein products for human use. As the biopharmaceutical industry looks forward to embracing new therapeutic modalities such as viral vector-mediated gene therapy, it is becoming evident that chromatographic separations will be also be crucial for success in that discipline. The current industry focus on cell culture intensification strategies that can result in increased process efficiency and lower cost of goods is presenting challenges to the robustness and economics of chromatography processes. To ensure robust and reproducible commercial manufacturing strategies, there is always a mandate to increase the scale of chromatography unit operations that are typically developed and optimized in small-scale development trials. This chapter discusses the key factors in typical chromatography operations that need to be carefully considered and modeled during the process scale-up phase in order to maintain the purity, yield, and quality of a product purified at smaller scales.

Sample Displacement Batch Chromatography of Proteins.

In downstream processing, large-scale chromatography plays an important role. For its development, screening experiments followed by pilot-plant chromatography are mandatory steps. Here we describe fast, simple, and inexpensive methods for establishing a preparative chromatography for the separation of complex protein mixtures, based on sample displacement batch chromatography. The methods are demonstrated by anion-exchange chromatography of a human plasma protein fraction (Cohn IV-4), including the screening step and upscaling of the chromatography by a factor of one hundred. The results of the screening experiments and the preparative chromatography are monitored by SDS-PAGE electrophoresis. In summary, we provide a protocol, which should be easily adaptable for the chromatographic large-scale purification of other proteins, in the laboratory as well as in the manufacturing of biopharmaceuticals. These protocols cover the initial piloting steps for establishing a large-scale sample batch chromatography. The results from the piloting steps may also be applied for packed columns for performing simulated-moving-bed (SMB) chromatography rather than batch chromatography.

The Application of Simulated Moving-Bed Chromatography for the Separation of Ginsenosides in Sanchi Ginseng and American Ginseng

In this study, the separation of ginsenosides from the crude extract of Sanchi ginseng and American ginseng, obtained by aqueous ethanol extraction, was conducted in a simulated moving bed (SMB) with cleaning in place (CIP) design. Concentrated products with 86.2%, w/w, and 91.5%, w/w, of ginsenosides were obtained by the utilization of CIP-SMB for Sanchi and American ginseng, respectively. This study reveals that CIP-SMB could enrich ginsenosides by removing the retained impurities, which were initially extracted by aqueous ethanol and not removed by either the antisolvent precipitation for Sanchi ginseng or the ethyl acetate extraction for American ginseng. Although the ginsenosides in both ginsengs are similar, the difficulty in applying chromatography to the crude extract differs. A natural crude extract normally contains compounds with significant difference in retention. A traditional SMB with 4 sections will be, consequently, unable to continuously separate the natural products. This study develops methods for pretreating the crude extract and demonstrates the feasibility of continuously separating ginsenosides by using SMB. SMB is an effective technology for large-scale chromatography and can generally resolve solvent and energy consumption problems. This is expected to promote the industrial use of SMB for the production of concentrated ginsenosides as active pharmaceutical ingredients.

Affinity purification of human alpha galactosidase utilizing a novel small molecule biomimetic of alpha-D-galactose

Alpha galactosidase (a-Gal) is an acidic hydrolase that plays a critical role in hydrolyzing the terminal alpha-galactoyl moiety from glycolipids and glycoproteins. There are over a hundred mutations reported for the GLA gene that encodes a-Gal that result in reduced protein synthesis, protein instability, and reduction in function. The deficiencies of a-Gal can cause Fabry disease, a rare lysosomal storage disorder (LSD) caused by the failure to catabolize alpha-d-galactoyl glycolipid moieties. The current standard of care for Fabry disease is enzyme replacement therapy (ERT) where the purified recombinant form of human a-Gal is given to patients. The manufacture of a-Gal is currently performed utilizing traditional large-scale chromatography processes. Developing an affinity resin for the purification of a-Gal would reduce the complexity of the manufacturing process, reduce costs, and potentially produce a higher quality a-Gal. After the evaluation of many small molecules, a commercially available small molecule biomimetic, N-5-Carboxypentyl-1-deoxygalactonojirimycin (N5C-DGJ), was utilized for the development of a novel small molecule biomimetic affinity resin for a-Gal. Affinity purified a-Gal demonstrated a purity greater than 90%, exhibited expected thermal stability and specific activity. Complementing this affinity purification is the development of an elution buffer system that confers an increased thermal stability to a-Gal. The N5C-DGJ affinity resin tolerated sodium hydroxide sanitization with no loss of binding capacity, making it amenable to large scale purification processes and potential use in manufacturing. This novel method for purifying the challenging a-Gal enzyme can be extended to other enzyme replacement therapies.

Recovery of sinapic acid from canola/rapeseed meal extracts by adsorption

Sinapic acid is a potential valuable compound to be recovered from rapeseed meal extracts as it processes antioxidant and antimicrobial properties. However, the concentration of this compound might be low and the presence of other low value compounds could complicate its downstream processing. Adsorption is an alternative technique that might allow selective recovery of this compound. This work was focused on establishing the foundation of an industrial process design to recover sinapic acid by adsorption. The obtained results from multicomponent experiments indicate that, resin AmberliteTM FPX66 is the best performing one showing a maximum adsorption capacity of 102.6+11.7mg/gresin, easy sinapic acid recovery by desorbing it with 70% ethanol and high selectivity to sinapic acid over glucose, phytic acid and glucosinolates. The obtained equilibrium information was applied as input in a dynamic column model and compared with experimental results, showing a good agreement (r2=0.98). The model can be further applied for a large-scale chromatography process design to recover sinapic acid from rapeseed/canola meal extracts.

A step-wise approach to define binding mechanisms of surrogate viral particles to multi-modal anion exchange resin in a single solute system.

Multi-modal anion exchange resins combine properties of both anion exchange and hydrophobic interaction chromatography for commercial protein polishing and may provide some viral clearance as well. From a regulatory viral clearance claim standpoint, it is unclear if multi-modal resins are truly orthogonal to either single-mode anion exchange or hydrophobic interaction columns. To answer this, a strategy of solute surface assays and High Throughput Screening of resin in concert with a scale-down model of large scale chromatography purification was employed to determine the predominant binding mechanisms of a panel of bacteriophage (i.e., PR772, PP7, and ϕX174) to multi-modal and single mode resins under various buffer conditions. The buffer conditions were restricted to buffer environments suggested by the manufacturer for the multi-modal resin. Each phage was examined for estimated net charge expression and relative hydrophobicity using chromatographic based methods. Overall, PP7 and PR772 bound to the multimodal resin via both anionic and hydrophobic moieties, while ϕX174 bound predominantly by the anionic moiety. Biotechnol. Bioeng. 2017;114: 1487-1494. © 2017 Wiley Periodicals, Inc.

Ambient Ionization: A Dream for the Surgical Oncologist?

One challenge for the cancer surgeon is to consistently distinguish tumor from healthy tissue and definitively remove the tumor with clean margins. The pathologist plays a key role in confirming the identity of the tissue removed, but it takes time to remove the sample, send it to the pathologist, and receive an expert analysis. In addition, this process is limited to the review of a small number of samples, given the burden on the pathologist. Is there a way for a surgeon to receive a near-real-time, intraoperative, automatic histological analysis? Dr. Graham Cooks, professor of analytical chemistry at Purdue University, thinks so. Desorption electrospray ionization-mass spectrometry (DESI-MS) ionizes samples without preparation and in the open environment. “The underlying principle is to reduce the amount of work that the analytical scientist does; in particular, reduce the grunt work regarding manipulation,” says Cooks in an interview with Clinical Chemistry . His passion for the subject came out of his PhD work, in which he conducted large-scale chromatography on the extensive plant material he was investigating. It took a year and …

Risk–benefit evaluation of on-line high-performance liquid chromatography analysis for pooling decisions in large-scale chromatography

In the production of a human therapeutic protein from inclusion bodies, product related impurities of very similar size and charge to the product are created as byproducts of the refold process. Their removal is usually challenging even when using chromatography with high performance resins and elution by shallow linear gradients. Additionally, performing this type of separation for commercial production adds increased complexity. To maximize productivity, columns are loaded so high that product elution profiles are not well separated from the impurities and pooling decisions are challenging. In this paper, conventional UV pooling based on fractionation or predefined absorbance based criteria will be compared to pooling based on fast on-line HPLC analytic. The development and implementation in a GMP process will be shown for a specific challenging separation by hydrophobic interaction chromatography. The different approaches have their unique complexities, timelines, uncertainties, and risks during development and implementation as well as during manufacturing. This study presents a probabilistic framework for quantitative comparison of two processes with unequal variability and uncertainty to evaluate the potential benefits of a PAT technology for its routine use in GMP Bioprocess manufacturing.

ChemInform Abstract: Large-Scale Chromatography in Industrial Processing.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Applied advanced process analytics in biopharmaceutical manufacturing: Challenges and prospects in real-time monitoring and control

Biopharmaceutical manufacturing processes are inherently complex due to their nonlinear bioprocess dynamics, variability in batch operations and manufacturing schedule, raw materials involved, and automatic process control. A typical processed lot generates large amounts of data that need to be analyzed and interpreted for process troubleshooting and continuous improvement purposes in addition to product release. Multivariate batch process modeling, monitoring and control approaches in real-time are elaborated by providing industrial examples from the commercial manufacturing processes. Examples and opportunities in cell culture (e.g., bioreactor applications) and purification (e.g., large-scale chromatography) operations are summarized. Impact of process analytical technologies (PAT), soft-sensor development, first principles modeling applications and commercial-scale examples are presented.

Applied Advanced Process Analytics in Biopharmaceutical Manufacturing: Challenges and Prospects in Real-time Monitoring and Control

Abstract Abstract Biopharmaceutical manufacturing processes are inherently complex due to their nonlinear bioprocess dynamics, variability in batch operations and manufacturing schedule, raw materials involved, and automatic process control. A typical processed lot generates large amounts of data that need to be analyzed and interpreted for process troubleshooting and continuous improvement purposes in addition to product release. Multivariate Batch Process Modeling, Monitoring and Control approaches in real-time are elaborated by providing industrial examples from the commercial manufacturing processes. Examples and opportunities in cell culture (e.g., bioreactor applications) and purification (e.g., large-scale chromatography) operations are summarized. Impact of Process Analytical Technologies (PAT), soft-sensor development, first principles modeling applications and commercial-scale examples are presented.

The fabrication of a microcolumn for gas separation using poly(dimethylsiloxane) as the structural and functional material

The fabrication and performance of a microcolumn made of poly(dimethylsiloxane) (PDMS) for gas separation is presented. PDMS is a material commonly used as a stationary phase in large-scale chromatography systems, while in this work it serves both as the structural material of the microcolumn as well as a stationary phase enabling gas mixture separation by different absorption and desorption kinetics of the sample components on the column walls. Gas separation is demonstrated for a xylene/benzene vapor mixture at different ratios detected by means of a VOC sensor connected at the exit of the microcolumn. The effect of the microcolumn depth and working temperature on the separation performance of the microcolumn is investigated.

Large-scale chromatography in industrial processing

Although chromatography has been established as a powerful analytical tool, its large-scale application is not widely appreciated. In this short review the up-to-date developments in the scaling-up of chromatographic processes are considered and the designs judged to offer the greatest potential and commercial viability are described paying special emphasis to liquid applications. Sufficient references are included to enable the interested reader to pursue the subject further.

Dry Column Vacuum Chromatography

Chromatographic purification is an integrated part of or- ganic synthesis. The Dry Column Vacuum Chromatography pre- sented here, has excellent resolving power, is easily applied to large scale chromatography (up to 100 g) and is fast. Furthermore, the technique is economical and environmentally friendly due to signif- icant reductions in solvent and the amount of silica used. Therefore, it is an excellent alternative to the commonly used Flash Column Chromatography for purification in organic synthesis.

Large-scale protein chromatography.

Large-scale protein chromatography.

Comparison of the efficacy of IGIV-C, 10% (caprylate/chromatography) and IGIV-SD, 10% as replacement therapy in primary immune deficiency: A randomized double-blind trial

A novel method of large-scale chromatography has been developed to improve recovery and purity of immunoglobulin G (IgG) from pooled plasma. The current study compares safety, toxicity and efficacy of two intravenous immunoglobulin products: a novel formulation, IGIV caprylate/chromatography (IGIV-C; Gamunex™, 10%) and a licensed solvent/detergent-treated product, Gamimune®N, 10% (IGIV-SD). The study, a randomized, double-blind, parallel group, therapeutic equivalence trial, was conducted at 25 treatment centers in Canada and the United States. Patients ( n=172) having confirmed chronic primary immunodeficiency (PID), aged 1–75 years, and receiving IGIV therapy were enrolled. For 9 months, patients were treated with IGIV-C or IGIV-SD in accordance with the patient's individualized treatment regimen utilized before study entry. The primary endpoint was the proportion of patients with ≥1 validated acute sinopulmonary infection during the treatment period. Secondary endpoints included the proportion of patients with all infections, time to first infection, annual infection rates, lung function parameters, infusion-related safety and viral safety. The annual validated infection rate in the IGIV-C group was 0.18 compared to 0.43 in the IGIV-SD group ( p=0.023). Nine patients receiving IGIV-C experienced validated infections, compared to 17 patients in IGIV-SD group ( p=0.06). Acute sinusitis (validated plus clinically defined) was less frequent in the IGIV-C group ( p=0.012). Presence of bronchiectasis did not affect efficacy. Adverse reactions were similar in frequency and severity in both groups. No evidence of viral transmission was observed. IGIV-C appears to be superior to IGIV-SD in preventing validated sinopulmonary infections, especially acute sinusitis, in patients with PID.

Evaluation of chromatographic recycling for imidazole used in the chromatographic purification of His-tag recombinant proteins

The aim of this work was to test a recycling method for imidazole used in immobilized metal affinity chromatography (IMAC) as eluent for recombinant histidine-tag (His-tag) protein. After evaluating two supports, the method was optimized with a mixture of bovine serum albumin, sodium chloride and imidazole. Recycling was performed with an eluate fraction from IMAC of His-tag enhanced green fluorescent protein produced in our laboratory and pure imidazole was recovered in water and was analyzed after being freeze-dried. The imidazole was then reused as eluent in IMAC without any modification in its structure or behavior. This procedure can be used for large-scale chromatography.

Oscillatory electroosmosis-enhanced intra/inter-particle liquid transport and its primary applications in the preparative electrochromatography of proteins

The concept of generating an oscillatory electroosmotic flux inside the porous particle to enhance the intra-particle mass transport was presented and a new kind of electrochromatography carried out in a five-compartment electrolyzer were developed. The adsorbent was packed in the central compartment, while the neighboring compartments were used as the elution compartments and the electrode compartments, respectively. Chromatographic separations of human serum albumin on Blue Sepharose Fast Flow, bovine serum albumin (BSA) on DEAE–Sepharose Fast Flow, and BSA on hydroxyapatite were carried out, respectively. The adsorption isotherms were shown to be independent of electric field, while the increase in the electric field strength resulted in a linear increase in the magnitude of electroosmotic flux and the improvement of the breakthrough behavior in all cases. The experiment results have demonstrated the effectiveness of the oscillatory electroosmosis in enhancing intra- and inter-particle mass transport and its high potential to large-scale chromatography.

Mass transport characteristics and chromatographic behavior of preparative electrochromatography on hydroxyapatite

Introducing an electric field into chromatography on hydroxyapatite (HAP) was attempted in order to enhance mass transfer and separation performance. A membrane spaced multicompartment electrolyzer was developed for electrochromatography on HAP. The high performance of liquid transport by electroosmotic flux was identified and described in terms of dynamic electroosmotic pressure. The application of the electric field resulted in an improved adsorption of bovine serum albumin as shown by the breakthrough curve as function of the electric field. An improved elution was also obtained in the presence of the electric field. The results show that electroosmosis is a powerful tool of liquid transport and dispersion in a packed bed of fine particles and has potential in the large-scale chromatography of biological molecules.

Optimization of large-scale chromatography of proteins

Protein chromatography is a very complex process based on a combination of thermodynamic, kinetic and mass transport phenomena. By virtue of their complicated and delicate surface structures, the behaviour of proteins on various Chromatographic media is not easy to predict. Together with the fact that the majority of the chromatographic media for proteins available today are not very well characterized with regard to their detailed chemical and physical surface structures, protein chromatography still has to be regarded as a predominantly empirical science. I.e. the optimization of separation conditions can only be performed by experiments in the laboratory. The scaling-up is then accomplished primarily by increasing the column diameter. This has been shown work well for column diameters up to at least 1,400 mm. The paper will also deal with the characteristics of the most important protein separation media, silica based, polystyrene based and agarose based, and with how to best optimize the conditions for column productivity, both for adsorption types of chromatography and for gel filtration.