Caprylic acid‐induced impurity precipitation from protein A capture column elution pool to enable a two‐chromatography‐step process for monoclonal antibody purification

This article presents the use of caprylic acid (CA) to precipitate impurities from the protein A capture column elution pool for the purification of monoclonal antibodies (mAbs) with the objective of developing a two chromatography step antibody purification process. A CA‐induced impurity precipitation in the protein A column elution pool was evaluated as an alternative method to polishing chromatography techniques for use in the purification of mAbs. Parameters including pH, CA concentrations, mixing time, mAb concentrations, buffer systems, and incubation temperatures were evaluated on their impacts on the impurity removal, high‐molecular weight (HMW) formation and precipitation step yield. Both pH and CA concentration, but not mAb concentrations and buffer systems, are key parameters that can affect host–cell proteins (HCPs) clearance, HMW species, and yield. CA precipitation removes HCPs and some HMW species to the acceptable levels under the optimal conditions. The CA precipitation process is robust at 15–25°C. For all five mAbs tested in this study, the optimal CA concentration range is 0.5–1.0%, while the pH range is from 5.0 to 6.0. A purification process using two chromatography steps (protein A capture column and ion exchange polishing column) in combination with CA‐based impurity precipitation step can be used as a robust downstream process for mAb molecules with a broad range of isoelectric points. Residual CA can be effectively removed by the subsequent polishing cation exchange chromatography. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1515–1525, 2015     

A practical strategy for using miniature chromatography columns in a standardized high‐throughput workflow for purification development of monoclonal antibodies

The emergence of monoclonal antibody (mAb) therapies has created a need for faster and more efficient bioprocess development strategies in order to meet timeline and material demands. In this work, a high‐throughput process development (HTPD) strategy implementing several high‐throughput chromatography purification techniques is described. Namely, batch incubations are used to scout feasible operating conditions, miniature columns are then used to determine separation of impurities, and, finally, a limited number of lab scale columns are tested to confirm the conditions identified using high‐throughput techniques and to provide a path toward large scale processing. This multistep approach builds upon previous HTPD work by combining, in a unique sequential fashion, the flexibility and throughput of batch incubations with the increased separation characteristics for the packed bed format of miniature columns. Additionally, in order to assess the applicability of using miniature columns in this workflow, transport considerations were compared with traditional lab scale columns, and performances were mapped for the two techniques. The high‐throughput strategy was utilized to determine optimal operating conditions with two different types of resins for a difficult separation of a mAb monomer from aggregates. Other more detailed prediction models are cited, but the intent of this work was to use high‐throughput strategies as a general guide for scaling and assessing operating space rather than as a precise model to exactly predict performance. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:626–635, 2014     

Multistage aqueous two‐phase extraction of a monoclonal antibody from cell supernatant

This article presents results of continuous multistage aqueous two‐phase extraction of an immunoglobulin G1 from cell supernatant in a mixer‐settler unit. An aqueous two‐phase system consisting of polyethylene glycol 2000, phosphate salt, and water was applied without and with sodium chloride (NaCl). Influences of different parameters such as throughput, phase ratio, and stage number on the extraction performance were analyzed. For systems without NaCl, the extraction was carried out as a washing step. An increase of stage number from one to five stages enabled to increase the immunoglobulin G1 purity from 11.8 to 32.6% at a yield of nearly 90%. Furthermore, a reduction of product phase volume due to a higher phase ratio led to an increase of purity from 20.8 to 29.6% in a three‐stage countercurrent extraction. For experiments with NaCl moderate partitioning conditions were adjusted by adding 8 wt% NaCl. In that case, the extraction was carried out as a stripping step. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:925–936, 2015     

Downstream processing of human antibodies integrating an extraction capture step and cation exchange chromatography

In this paper we explore an alternative process for the purification of human antibodies from a Chinese hamster ovary (CHO) cell supernatant comprising a ligand-enhanced extraction capture step and cation exchange chromatography (CEX). The extraction of human antibodies was performed in an aqueous two-phase system (ATPS) composed of dextran and polyethylene glycol (PEG), in which the terminal hydroxyl groups of the PEG molecule were modified with an amino acid mimetic ligand in order to enhance the partition of the antibodies to the PEG-rich phase. This capture step was optimized using a design of experiments and a central composite design allowed the determination of the conditions that favor the partition of the antibodies to the phase containing the PEG diglutaric acid (PEG-GA) polymer, in terms of system composition. Accordingly, higher recovery yields were obtained for higher concentrations of PEG-GA and lower concentrations of dextran. The highest yield experimentally obtained was observed for an ATPS composed of 5.17% (w/w) dextran and 8% (w/w) PEG-GA. Higher purities were however predicted for higher concentrations of both polymers. A compromise between yield and purity was achieved using 5% dextran and 10% PEG-GA, which allowed the recovery of 82% of the antibodies with a protein purity of 96% and a total purity of 63%, determined by size-exclusion chromatography. ATPS top phases were further purified by cation exchange chromatography and it was observed that the most adequate cation exchange ligand was carboxymethyl, as the sulfopropyl ligand induced the formation of multi-aggregates or denatured forms. This column allowed the elution of 89% of the antibodies present in the top phase, with a protein purity of 100% and a total purity of 91%. The overall process containing a ligand-enhanced extraction step and a cation exchange chromatography step had an overall yield of 73%.     

以聚乙二醇为层析伴侣同时制备SOD、过氧化氢酶和血红蛋白

发展了一条从红细胞裂解液中同时制备超氧化物歧化酶(SOD)、过氧化氢酶和血红蛋白的新工艺。采用0 75 %的聚乙二醇600作为层析伴侣,使血红蛋白直接流过阴离子交换层析柱,同时吸附SOD和过氧化氢酶。经过梯度洗脱获得SOD和过氧化氢酶组分,再经过疏水性相互作用层析与凝胶过滤层析相串联,使SOD和过氧化氢酶得到纯化。纯化后的SOD和过氧化氢酶的比活力分别达到15932u/mg和65918u/mg ,血红蛋白的纯度达到99.9%以上。总回收率为:SOD ,47.4% ;过氧化氢酶,29.6% ;血红蛋白,88.7%。     

Rapid purification of EGFP, EYFP, and ECFP with high yield and purity

Most current high throughput purification procedures for the green fluorescent protein (GFP) suffer from poor yields and low purity. An improved purification procedure that delivers highly pure protein (>95% homogeneity) in high yields (>70% of the initial fluorescent protein content) has been developed. The purification procedure requires only two steps: the cell lysate is heated to 60 degrees C for 4 min in ammonium sulfate and triethylamine, followed by hydrophobic interaction chromatography using isopropanol during the elution phase. The resulting pure product exhibits the same fluorescence profile as the crude sample. This procedure has been demonstrated on three commercial variants of GFP from Aequorea victoria, enhanced green, enhanced yellow, and enhanced cyan fluorescent protein (Becton-Dickinson). The yield and purity of material are superior to other recently described methods.     

Purification and characterization of the gag gene products of avian-type C retroviruses by high-pressure liquid chromatography

We have developed an inexpensive procedure for bacteriophage purification suitable for small- and medium-scale preparations (up to one liter of lysate). The method consists of precipitation with polyethylene glycol 6000 and gel chromatography on a Bio-Gel A-5m column. The purity of the phage preparation is comparable to that obtained by CsCl step gradient ultracentrifugation.     

Purification of equine chorionic gonadotropin (eCG) using magnetic ion exchange adsorbents in combination with high‐gradient magnetic separation

Current purification of the glycoprotein equine chorionic gonadotropin (eCG) from horse serum includes consecutive precipitation steps beginning with metaphosphoric acid pH fractionation, two ethanol precipitation steps, and dialysis followed by a numerous of fixed‐bed chromatography steps up to the specific activity required. A promising procedure for a more economic purification procedure represents a simplified precipitation process requiring only onethird of the solvent, followed by the usage of magnetic ion exchange adsorbents employed together with a newly designed ‘rotor‐stator’ type High Gradient Magnetic Fishing (HGMF) system for large‐scale application, currently up to 100 g of magnetic adsorbents. Initially, the separation process design was optimized for binding and elution conditions for the target protein in mL scale. Subsequently, the magnetic filter for particle separation was characterized. Based on these results, a purification process for eCG was designed consisting of (i) pretreatment of the horse serum; (ii) binding of the target protein to magnetic ion exchange adsorbents in a batch reactor; (iii) recovery of loaded functionalized adsorbents from the pretreated solution using HGMF; (iv) washing of loaded adsorbents to remove unbound proteins; (v) elution of the target protein. Finally, the complete HGMF process was automated and conducted with either multiple single‐cycles or multicycle operation of four sequential cycles, using batches of pretreated serum of up to 20 L. eCG purification with yields of approximately 53% from single HGMF cycles and up to 80% from multicycle experiments were reached, with purification and concentration factors of around 2,500 and 6.7, respectively. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:78–89, 2015     

A label-free methodology for selective protein quantification by means of absorption measurements

The application of high throughput experimentation (HTE) in protein purification process development has created an analytical bottleneck. Using a new label-free and non-invasive methodology for analyzing multicomponent protein mixtures by means of spectral measurements, we show that the analytical throughput for selective protein quantification can be increased significantly. An analytical assay based on this new methodology was shown to generate very precise results. Further, the assay was successfully applied as analytics for a resin screening performed in HTE mode. The increase in analytical throughput was obtained without decreasing the level of information when compared to analytical chromatography. This proves its potential as a valuable analytical tool in conjugation with high throughput process development (HTPD). Further, fast selective protein quantification can enhance process control in a commercial production environment and, hence, minimize the need for off-line release analysis.     

Chromatography process development in the quality by design paradigm I: Establishing a high‐throughput process development platform as a tool for estimating “characterization space” for an ion exchange chromatography step

This article describes the development of a high‐throughput process development (HTPD) platform for developing chromatography steps. An assessment of the platform as a tool for establishing the “characterization space” for an ion exchange chromatography step has been performed by using design of experiments. Case studies involving use of a biotech therapeutic, granulocyte colony‐stimulating factor have been used to demonstrate the performance of the platform. We discuss the various challenges that arise when working at such small volumes along with the solutions that we propose to alleviate these challenges to make the HTPD data suitable for empirical modeling. Further, we have also validated the scalability of this platform by comparing the results from the HTPD platform (2 and 6 μL resin volumes) against those obtained at the traditional laboratory scale (resin volume, 0.5 mL). We find that after integration of the proposed correction factors, the HTPD platform is capable of performing the process optimization studies at 170‐fold higher productivity. The platform is capable of providing semi‐quantitative assessment of the effects of the various input parameters under consideration. We think that platform such as the one presented is an excellent tool for examining the “characterization space” and reducing the extensive experimentation at the traditional lab scale that is otherwise required for establishing the “design space.” Thus, this platform will specifically aid in successful implementation of quality by design in biotech process development. This is especially significant in view of the constraints with respect to time and resources that the biopharma industry faces today. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 403–414, 2013     

Strategy for a protein purification design using C-phycocyanin extract

A variety of techniques have been developed for the separation and recovery of proteins. The cost of purifying the product is frequently determined by the desired quality of the final product, which is evaluated by measuring the purity. In this work the design of a protein purification process for C-phycocyanin, a phycobiliprotein that can be used in the food and medical industries, was established. The study evaluated the use of ammonium sulfate precipitation, ion exchange chromatography and gel filtration to purify C-phycocyanin in a variety of sequences. The final design included the C-phycocyanin extraction step, precipitation with ammonium sulfate and ion exchange chromatography. When the elution step was studied, the kind of elution and pH were considered in order to obtain a product with a final purity of 4.0 with a purification factor of 6.35, so that, at the end of the strategy, C-phycocyanin of analytical grade would be obtained.     

High throughput chromatography and analytics can inform viral clearance capabilities during downstream process development for biologics

High throughput process development (HTPD) using liquid handling robotics and RoboColumns is an established methodology in downstream process development to screen chromatography resins and optimize process designs to meet target product profiles. However, HTPD is not yet widely available for use in viral clearance capability of the resin due to a variety of constraints. In the present study, a BSL-1-compatible, non-infectious MVM model, MVM-VLP, was tested for viral clearance assessment with various resin and membrane chromatography operations in a HTPD mode. To detect the MVM-VLP in the high throughput experiments, an electrochemiluminescence immunoassay (ECLIA) assay was developed with up to 5 logs of dynamic range. Storage time suitability of MVM-VLP solutions in various buffer matrices, in the presence or absence of a glycoprotein vaccine candidate, were assessed. Then, MVM-VLP and a test article monoclonal antibody (mAb) were used in a HTPD design that included commercially available ion exchange media chemistries, elucidating a wide variety of viral clearance ability at different operating conditions. The methodologies described herein have the potential to be a part of the process design stage in biologics manufacturing process development, which in turn can reduce risk associated with viral clearance validation studies.     

Cation exchange as a single polishing step for conjugated peptides

Purification of peptides typically includes expensive reverse phase (RP) processes, which utilize high pressure and large volumes of solvent. For two conjugated peptides, chromatography process development targeted a low-pressure aqueous process that could achieve target product purities of ≥95%, comparable to purities seen with traditional RP. A high throughput screening approach of different modalities was used to identify binding and elution conditions on a cation exchange resin and small-scale columns were used in order to assess impurity removal and process yield. The parameters for load and gradient elution were optimized to increase product purity and process productivity with a wide operating window identified where high purity and productivity are achieved. Computational modeling was then used to validate experimental chromatography results and to gain insight on the effect of the chemical modifications on the surface properties of the two peptides. Both modeling and experimental data showed that with optimization, cation exchange could be utilized as a single polishing step for conjugated peptides. Similar purities were achieved as those seen with RP with up to double the productivity.     

A nonchromatographic process for purification of secretory immunoglobulins from caprine whey

Secretory immunoglobulins are an important antibody class being primarily responsible for immunoprotection of mucosal surfaces. A simple, non‐chromatographic purification process for secretory immunoglobulins from caprine whey was developed. In the first process step whey was concentrated 30–40‐fold on a 500 kDa membrane, thereby increasing the purity from 3% to 15%. The second step consisted of a fractionated PEG precipitation, in which high molecular weight impurities were removed first and in the second stage the secretory immunoglobulins were precipitated, leaving a majority of the low molecular weight proteins in solution. The re‐dissolved secretory immunoglobulin fraction had a purity of 43% which could then be increased to 72% by diafiltration at a volume exchange factor of 10. Further increase of purity was only possible at the expense of very high buffer consumption. If diafiltration was performed directly after ultrafiltration, followed by precipitation, the yield was higher but purity was only 54%. Overall, filtration performance was characterized by high concentration polarization, therefore process conditions were set to low trans‐membrane pressure and moderate protein concentration. As such purity and to a lesser extent throughput were the major objectives rather than yield, since whey, as a by‐product of the dairy industry, is a cheap raw material of almost unlimited supply. Ultra‐/diafiltration performance was described well by correlations using dimensionless numbers. Compared with a theoretical model (Graetz/Leveque solution) the flux was slightly overestimated. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:642–653, 2017     

Clarification and capture of high‐concentration refold pools for E. coli‐based therapeutics using expanded bed adsorption chromatography

Expanded bed adsorption (EBA) chromatography was investigated for clarification and capture of high‐concentration refold pools of Escherichia coli‐based therapeutics. Refolding of denatured inclusion bodies (IBs) at high protein concentration significantly improved product throughput; however, direct filtration of the refold materials became very challenging because of high content of protein precipitates formed during refolding. In addition, irreversible protein precipitation caused by high local concentration was encountered in packed bed capture during cation exchange chromatography elution, which limited column loading capacity and capture step productivity. In this study, the two issues are addressed in one unit operation by using EBA. Specifically, EBA can handle feed streams with significant amount of particles and precipitates, which eliminated the need for refold pool clarification through filtration. The relatively broad EBA elution profile is particularly suitable for proteins of low solubility and can effectively avoid product loss previously associated with on‐column precipitation during capture. As the EBA resin (RHOBUST^® FastLine SP IEX) used here has unique properties, it can be operated at high linear velocity (800–1,600 cm/h), while achieving a selectivity and impurity clearance largely comparable to the packed bed resin of the same ligand chemistry (SP Sepharose FF). Furthermore, the filtration of the EBA elution pool is easily manageable within facility capability. Overall, this study demonstrates that the EBA process helps debottleneck the purification of high‐turbidity refold pools by removing precipitates and concurrently capturing the product, which can be applied to other E. coli‐based therapeutics that also requires refolding of IBs. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:113–123, 2014     

Single-step purification of a small non-mAb biologic by peptide-ELP-based affinity precipitation

Affinity precipitation using stimulus-responsive biopolymers such as elastin-like polypeptides (ELPs) have been successfully employed for the purification of monoclonal antibodies. In the current work, we extend these studies to the development of an ELP-peptide fusion for the affinity precipitation of the therapeutically relevant small non-mAb biologic, AdP. A 12-mer affinity peptide ligand (P10) was identified by a primary phage biopanning followed by a secondary in-solution fluorescence polarization screen. Peptide P10 and AdP interacted with a K_D of 19.5 µM. A fusion of P10 with ELP was then shown to be successful in selectively capturing the biologic from a crude mixture. While pH shifts alone were not sufficient for product elution, the use of pH in concert with fluid-phase modifiers such as NaCl, arginine, or ethylene glycol was effective. In particular, the use of pH 8.5 and an arginine concentration of 500 mM enabled >80% product recovery. The overall process performance evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase ultra-performance liquid chromatography analyses indicated successful single-step purification of the biologic from an Escherichia coli lysate resulting in ∼90% purity and >80% recovery. These results demonstrate that phage display can be readily employed to identify a peptide ligand capable of successfully carrying out the purification of a non-antibody biological product using ELP-based affinity precipitation.     

An Integrated Approach to Aggregate Control for Therapeutic Bispecific Antibodies Using an Improved Three Column Mab Platform-Like Purification Process

Single chain variable fragment-IgGs (scFv-IgG) are a class of bispecific antibodies consisting of two single chain variable fragments (scFv) that are fused to an intact IgG molecule. A common trend observed for expression of scFv-IgGs in mammalian cell culture is a higher level of aggregates (10%–30%) compared to mAbs, which results in lower purification yields in order to meet product quality targets. Furthermore, the high aggregate levels also pose robustness risks to a conventional mAb three column platform purification process which uses only the polishing steps (e.g., cation exchange chromatography [CEX]) for aggregate removal. Protein A chromatography with pH gradient elution, high performance tangential flow filtration (HP-TFF) and calcium phosphate precipitation were evaluated at the bench scale as means of introducing orthogonal aggregate removal capabilities into other aspects of the purification process. The two most promising process variants, namely Protein A pH gradient elution followed by calcium phosphate precipitation were evaluated at pilot scale, demonstrating comparable performance. Implementing Protein A chromatography with gradient elution and/or calcium phosphate precipitation removed a sufficient portion of the aggregate burden prior to the CEX polishing step, enabling CEX to be operated robustly under conditions favoring higher monomer yield. From starting aggregate levels ranging from 15% to 23% in the condition media, levels were reduced to between 2% and 3% at the end of the CEX step. The overall yield for the optimal process was 71%. Results of this work suggest an improved three-column mAb platform-like purification process for purification of high aggregate scFv-IgG bispecific antibodies is feasible. © 2018 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers. Biotechnol. Prog., 35: e2720, 2019     

Resolution of heterogeneous charged antibody aggregates via multimodal chromatography: A comparison to conventional approaches

Clearance of aggregates during protein purification is increasingly paramount as protein aggregates represent one of the major impurities in biopharmaceutical products. Aggregates, especially dimer species, represent a significant challenge for purification processing since aggregate separation coupled with high purity protein recovery can be difficult to accomplish. Biochemical characterization of the aggregate species from the hydrophobic interaction and cation exchange chromatography elution peaks revealed two different charged populations, i.e. heterogeneous charged aggregates, which led to further challenges for chromatographic removal. This paper compares multimodal versus conventional cation exchange or hydrophobic chromatography methodologies to remove heterogeneous aggregates. A full, mixed level factorial design of experiment strategy together with high throughput experimentation was employed to rapidly evaluate chromatographic parameters such as pH, conductivity, and loading. A variety of operating conditions were identified for the multimodal chromatography step, which lead to effective removal of two different charged populations of aggregate species. This multimodal chromatography step was incorporated into a monoclonal antibody purification process and successfully implemented at commercial manufacturing scale. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:636–645, 2014     

Purification and properties of rat brain pyruvate carboxylase.

Rat brain pyruvate carboxylase was purified 2000-fold and some of its properties and kinetic parameters were investigated. The use of (NH4)2SO4 gradient solubilization on a Celite column and precipitation with polyethylene glycol permitted purification to an estimated 20% purity. Except for a few subtle kinetic differences this enzyme is indistinguishable from rat liver pyruvate carboxylase.     

Downstream processing of extracellular phytase from Aspergillus niger: Chromatography process vs. aqueous two phase extraction for its simultaneous partitioning and purification

The application of single step aqueous two-phase extraction (ATPE) for the downstream processing of phytase from Aspergillus niger NCIM 563, produced under solid state fermentation, has been studied and compared with the traditional multi-step procedure involving salt precipitation and column chromatography. High phytase recovery (98.5%) within a short time (3 h) and improved thermostability was attained by ATPE in comparison to 20% recovery in 96 h by chromatography process. The ATPE method, therefore, seems to be an interesting alternative for simultaneous partitioning and purification of phytase. The influence of system parameters; such as, phase forming salts, polymer molecular weight and system pH on the partitioning behavior of phytase was evaluated. The ATPE system consisting of combination of polyethylene glycol (PEG) 6000 and 8000 (10.5%) and sodium citrate (20.5%) resulted in one-sided partitioning of phytase in bottom phase with a purification factor of 2.5. This is the first report on phytase purification using liquid–liquid extraction and the results are likely to be beneficial in the poultry feed industry.