Downstream antibody purification using aqueous two‐phase extraction

The extraction of antibodies using a polyethylene glycol (PEG)‐citrate aqueous two‐phase system (ATPS) was investigated. Studies using purified monoclonal antibody (mAb) identified operating ranges for successful phase formation and factors that significantly affected antibody partitioning. The separation of antibody and host cell protein (HCP) from clarified cell culture media was examined using statistical design of experiments (DOE). The partitioning of antibody was nearly complete over the entire range of the operating space examined. A model of the HCP partitioning was generated in which both NaCl and citrate concentrations were identified as significant factors. To achieve the highest purity, the partitioning of HCP from cell culture fluid into the product containing phase was minimized using a Steepest Descent algorithm. An optimal ATPS consisting of 14.0% (w/w) PEG, 8.4% (w/w) citrate, and 7.2% (w/w) NaCl at pH 7.2 resulted in a product yield of 89%, an approximate 7.6‐fold reduction in HCP levels relative to the clarified cell culture fluid before extraction and an overall purity of 70%. A system consisting of 15% (w/w) PEG, 8% (w/w) citrate, and 15% (w/w) NaCl at pH 5.5 reduced product‐related impurities (aggregates and low molecular product fragments) from ～40% to less than 0.5% while achieving 95% product recovery. At the experimental conditions that were optimized in the batch mode, a scale‐up model for the use of counter‐current extraction technology was developed to identify potential improvements in purity and recovery that could be realized in the continuous operational mode. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Influence of osmolarity and pH increase to achieve a reduction of monoclonal antibodies aggregates in a production process

Anti PSA monoclonal antibodies for diagnostic use were produced in an in vitro system. After purification using Protein G affinity chromatography a percentage of about 10% of antibody aggregates remained. The use of monoclonal antibodies containing aggregates as a capture antibody in a diagnostic kit reduces the performance of the test making it often unacceptable. The aggregates could be eliminated using gel filtration chromatography but, in that way, the final recovery of the whole production process was only about 50%. Aggregation is favoured when the working pH is near to the isoelectric point of the antibody. We varied the culture medium composition, modifying pH and osmolarity. We tested different values of pH and osmolarity: 7.1, 7.5, 8.0, 8.5 for pH, and 300, 340, 367, 395 mOsm/kg H2O for osmolarity. By modification of the cell culture medium we obtained a significant decrease of monoclonal antibody aggregates in the production cycle. In this way we achieved higher recovery rate and could avoid gel filtration polishing step. The experiments were performed in two stages: first in culture flasks changing one parameter in each experiment, and then in spinner bottle using the best conditions obtained in the first stage. During scale up we used the modifications achieved from the experiment showed in this paper in our production by hollow fibre bioreactor with positive results.     

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     

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     

Selective antibody precipitation using polyelectrolytes: A novel approach to the purification of monoclonal antibodies

We evaluated the potential for polyelectrolyte induced precipitation of antibodies to replace traditional chromatography purification. We investigated the impact of solution pH, solution ionic strength and polyelectrolyte molecular weight on the degree of precipitation using the anionic polyelectrolytes polyvinylsulfonic acid (PVS), polyacrylic acid (PAA), and polystyrenesulfonic acid (PSS). As we approached the pI of the antibody, charge neutralization of the antibody reduced the antibody–polyelectrolyte interaction, reducing antibody precipitation. At a given pH, increasing solution ionic strength prevented the ionic interaction between the polyelectrolyte and the antibody, reducing antibody precipitation. With increasing pH of precipitation, there was an increase in impurity clearance. Increasing polyelectrolyte molecular weight allowed the precipitation to be performed under conditions of higher ionic strength. PVS was selected as the preferred polyelectrolyte based on step yield following resolubilization, purification performance, as well as the nature of the precipitate. We evaluated PVS precipitation as a replacement for the initial capture step, as well as an intermediate polishing step in the purification of a humanized monoclonal antibody. PVS precipitation separated the antibody from host cell impurities such as host cell proteins (HCP) and DNA, process impurities such as leached protein A, insulin and gentamicin, as well as antibody fragments and aggregates. PVS was subsequently removed from antibody pools to <1 µg/mg using anion exchange chromatography. PVS precipitation did not impact the biological activity of the resolubilized antibody. Biotechnol. Bioeng. 2009;102: 1141–1151. © 2008 Wiley Periodicals, Inc.     

Monoclonal antibody purification using cationic polyelectrolytes: an alternative to column chromatography

The potential of cationic polyelectrolytes to precipitate host cell and process related impurities was investigated, to replace one or more chromatography steps in monoclonal antibody purification. The impact of antibody isoelectric point, solution properties (pH and ionic strength), and polyelectrolyte properties (structure, molecular weight and pK(a)) on the degree of precipitation was studied. At neutral pH, increasing solution ionic strength impeded the ionic interaction between the polyelectrolyte and impurities, reducing impurity precipitation. Increasing polyelectrolyte molecular weight and pK(a) enabled precipitation of impurities at higher ionic strength. PoIy(arginine) was selected as the preferred polyelectrolyte in unconditioned cell culture fluid. PoIy(arginine) precipitation achieved consistent host cell protein clearance and antibody recovery for multiple antibodies across a wider range of polyelectrolyte concentrations. Poly(arginine) precipitation was evaluated as a flocculant and as a functional replacement for anion exchange chromatography in an antibody purification process. Upstream treatment of cell culture fluid with poly(arginine) resulted in flocculation of solids (cells and cell debris), and antibody recovery and impurity clearance (host cell proteins, DNA and insulin) comparable to the downstream anion exchange chromatography step.     

An integrated practical implementation of continuous aqueous two‐phase systems for the recovery of human IgG: From the microdevice to a multistage bench‐scale mixer‐settler device

Aqueous two‐phase systems (ATPS) are a liquid‐liquid extraction technology with clear process benefits; however, its lack of industrial embracement is still a challenge to overcome. Antibodies are a potential product to be recovered by ATPS in a commercial context. The objective of this work is to present a more integral approach of the different isolated strategies that have arisen in order to enable a practical, generic implementation of ATPS, using human immunoglobulin G (IgG) as experimental model. A microfluidic device is used for ATPS parameters preselection for product recovery. ATPS were continuously operated in a mixer‐settler device in one stage, multistage and multistage with recirculation configuration. Single‐stage pure IgG extraction with a polyethylene glycol (PEG) 3350‐phophates ATPS within continuous operation allowed a 65% recovery. Further implementation of a multistage platform promoted a higher particle partitioning reaching a 90% recovery. The processing of IgG from a cell supernatant culture harvest in a multistage system with top phase recirculation resulted in 78% IgG recovery in bottom phase. This work conjugates three not widely spread methodologies for ATPS: microfluidics, continuous and multistage operation.     

An impurity characterization based approach for the rapid development of integrated downstream purification processes

In this study, we describe a new approach for the characterization of process‐related impurities along with an in silico tool to generate orthogonal, integrated downstream purification processes for biological products. A one‐time characterization of process‐related impurities from product expression in Pichia pastoris was first carried out using linear salt and pH gradients on a library of multimodal, salt‐tolerant, and hydrophobic charge induction chromatographic resins. The Reversed‐phase ultra‐performance liquid chromatography (UPLC) analysis of the fractions from these gradients was then used to generate large data sets of impurity profiles. A retention database of the biological product was also generated using the same linear salt and pH gradients on these resins, without fraction collection. The resulting two data sets were then analyzed using an in silico tool, which incorporated integrated manufacturing constraints to generate and rank potential three‐step purification sequences based on their predicted purification performance as well as whole‐process “orthogonality” for impurity removal. Highly ranked sequences were further examined to identify templates for process development. The efficacy of this approach was successfully demonstrated for the rapid development of robust integrated processes for human growth hormone and granulocyte‐colony stimulating factor.     

Affinity partitioning with polymer-bound Cibacron blue F3G-A for rapid,large-scale purification of phosphofructokinase from baker's yeast

Phosphofructokinase has been isolated in homogenous form from baker's yeast. The first two steps, fractional precipitation with polyethylene glycol and affinity partitioning in aqueous biphasic systems containing Cibacron blue F3G-A-polyethylene glycol, gave a 58-fold purification within 3 h. In these steps the amount of contaminating proteases was reduced by 2 orders of magnitude. After concentration using DEAE-cellulose followed by gel chromatography, homogeneous enzyme was obtained. The advantages of affinity partitioning for large-scale preparations are discussed.     

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     

High‐efficiency affinity precipitation of multiple industrial mAbs and Fc‐fusion proteins from cell culture harvests using Z‐ELP‐E2 nanocages

Affinity precipitation using Z‐elastin‐like polypeptide‐functionalized E2 protein nanocages has been shown to be a promising alternative to Protein A chromatography for monoclonal antibody (mAb) purification. We have previously described a high‐yielding, affinity precipitation process capable of rapidly capturing mAbs from cell culture through spontaneous, multivalent crosslinking into large aggregates. To challenge the capabilities of this technology, nanocage affinity precipitation was investigated using four industrial mAbs (mAbs A–D) and one Fc fusion protein (Fc A) with diverse molecular properties. A molar binding ratio of 3:1 Z:mAb was sufficient to precipitate >95% mAb in solution for all molecules evaluated at ambient temperature without added salt. The effect of solution pH on aggregation kinetics was studied using a simplified two‐step model to investigate the protein interactions that occur during mAb–nanocage crosslinking and to determine the optimal solution pH for precipitation. After centrifugation, the pelleted mAb–nanocage complex remained insoluble and was capable of being washed at pH ≥ 5 and eluted with at pH < 4 with >90% mAb recovery for all molecules. The four mAbs and one Fc fusion were purified from cell culture using optimal process conditions, and >94% yield and >97% monomer content were obtained. mAb A–D purification resulted in a 99.9% reduction in host cell protein and >99.99% reduction in DNA from the cell culture fluids. Nanocage affinity precipitation was equivalent to or exceeded expected Protein A chromatography performance. This study highlights the benefits of nanoparticle crosslinking for enhanced affinity capture and presents a robust platform that can be applied to any target mAb or Fc‐containing proteins with minimal optimization of process parameters.     

Transformation of ferulic acid to vanillin using a fed‐batch solid–liquid two‐phase partitioning bioreactor

Amycolatopsis sp. ATCC 39116 (formerly Streptomyces setonii) has shown promising results in converting ferulic acid (trans‐4‐hydroxy‐3‐methoxycinnamic acid; substrate), which can be derived from natural plant wastes, to vanillin (4‐hydroxy‐3‐methoxybenzaldehyde). After exploring the influence of adding vanillin at different times during the growth cycle on cell growth and transformation performance of this strain and demonstrating the inhibitory effect of vanillin, a solid–liquid two‐phase partitioning bioreactor (TPPB) system was used as an in situ product removal technique to enhance transformation productivity by this strain. The thermoplastic polymer Hytrel^® G4078W was found to have superior partitioning capacity for vanillin with a partition coefficient of 12 and a low affinity for the substrate. A 3‐L working volume solid–liquid fed‐batch TPPB mode, using 300 g Hytrel G4078W as the sequestering phase, produced a final vanillin concentration of 19.5 g/L. The overall productivity of this reactor system was 450 mg/L. h, among the highest reported in literature. Vanillin was easily and quantitatively recovered from the polymers mostly by single stage extraction into methanol or other organic solvents used in food industry, simultaneously regenerating polymer beads for reuse. A polymer–liquid two phase bioreactor was again confirmed to easily outperform single phase systems that feature inhibitory or easily further degraded substrates/products. This enhancement strategy might reasonably be expected in the production of other flavor and fragrance compounds obtained by biotransformations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:207–214, 2014     

An Integrated System for the Bioremediation of Wastewater Containing Xenobiotics and Toxcic Metals

An integrated system for the biotreatment of acidic wastewaters containing both toxic metals and organics is presented. It consists of two bioprocess stages (i) an anaerobic, SRB stage (containing alkaline‐tolerant s ulfate‐ r educing b acteria) that at pH 8 (chosen to acclimatize the bacteria in the biomedium) produces high concentrations of total sulfide ions (more than 400 mg/L) which are added to the wastewater to precipitate the heavy metals out at pH 2 as metal sulfides, and (ii) an aerobic, acidophilic stage containing heterotrophic bacteria (WJB3) that degrade organic xenobiotics. The anaerobic system was comprised of a 4‐L fluidized bed bioreactor with immobilized SRB, a mixing tank, and a precipitation tank. The effluent from the bioreactor with a high concentration of sulfide ions was fed into a mixing tank where model wastewaters containing toxic metals and phenol at pH 2 were also fed at increasing loading rates until free metal ions could be detected in the precipitation tank outlet. Then the effluent from the precipitation tank outlet was fed into a 2.5‐L aerobic bioreactor in which phenol was degraded. In this research, 100 % removal efficiencies were obtained with wastewaters containing more than 400 mg/L metal ions and 900 mg/L phenol at a 6‐h HRT of the mixing tank.     

Using precipitation by polyamines as an alternative to chromatographic separation in antibody purification processes

Polyamine precipitation conditions for removing host cell protein impurities from the cell culture fluid containing monoclonal antibody were studied. We examined the impact of polyamine concentration, size, structure, cell culture fluid pH and ionic strength. A 96-well microtiter plate based high throughput screening method was developed and used for evaluating different polyamines. Polyallylamine, polyvinylamine, branched polyethyleneimine and poly(dimethylamine-co-epichlorohydrin-ethylenediamine) were identified as efficient precipitants in removing host cell protein impurities. Leveraging from the screening results, we incorporated a polyamine precipitation step into a monoclonal antibody purification process to replace the Protein A chromatography step. The optimization of the overall purification process was performed by taking the mechanisms of both precipitation and chromatographic separation into account. The precipitation-containing process removed a similar amount of process-related impurities, including host cell proteins, DNA, insulin and gentamicin and maintained similar product quality in respect of size and charge variants to chromatography based purification. Overall recovery yield was comparable to the typical Protein A affinity chromatography based antibody purification process.     

固定化胰蛋白酶亲和层析法制备低STI残存大豆分离蛋白质

聚苯乙烯阴离子交换树脂(GM201)经预处理除去杂质后先与戊二醛(2—6％)反应,再与胰蛋白酶(5000u/mg,8—10mg/mL,pH 8.0)反应即制得固定化胰蛋白酶。此法得到的固定化胰蛋白酶具有良好的热稳定性,贮藏稳定性和操作稳定性,可用于工业化目的。脱脂豆粉经萃取(PH9.0)后,稀释4倍,在pH5.0下沉淀分离出大豆球蛋白,然后用酸性水(pH5.0)洗涤两次,并进行碱溶与酸沉淀两次,即可将大豆分离蛋白质的STI残留降低到1.85％,比活性降到1u/mg以下。最后再用固定化胰蛋白酶亲和层析,就可以除去大豆分离蛋白质中残留的STI。     

Purification of protein C from human plasma by precipitation and aqueous two-phase partitioning

Summary Precipitation with PEG and partitioning in PEG/dextran aqueous two-phase systems are applied to the purification of protein C, a human plasma protein with a key role in anti-coagulation. Tandem application of precipitation followed by partitioning yields a purification factor for protein C greater than that using either process individually. The results are discussed using the hypothesis that the mechanism of solvation of protein C by PEG is similar, while that for total plasma proteins is different, in the two processes.     

Quantitative comparison on the refinement of horse antivenom by salt fractionation and ion-exchange chromatography

A quantitative comparison was made on the fractionation of pepsin-digested horse antivenoms by ammonium sulfate (AS) fractional precipitation and ion-exchange chromatography on Q-Sepharose. In the precipitation process, pepsin digested horse anti-Naja kaouthia serum was precipitated by 30% saturated AS followed by 50% saturated AS. The recovery of antibody activity [as measured by an enzyme-linked immunosorbent assay (ELISA) against the cobra postsynaptic neurotoxin 3] from the 30–50% saturated AS precipitate was 53% with a 1.93-fold purification. For the chromatographic process, the behavior of the horse antitoxin antibody and its F(ab′)₂ fragments was first studied. The pepsin digested horse serum was then desalted on a Bio-gel P-2 column followed by chromatography on Q-Sepharose using a linear gradient (20 mM Tris-HCl, pH 8.0 containing 0.0 to 0.5 M NaCl). A peak containing primarily the F(ab′)₂ antibody could be obtained. This peak constituted 73% of the total antivenom activity with 2.08-fold purification. The total recovery of antibody activity by the chromatographic process was 90%. The yield of antibody activity was about 2-fold higher than that reported previously with other fractionation procedures. The implications of these results for the refining of horse therapeutic antivenoms are discussed.     

Purification process development of a recombinant monoclonal antibody expressed in glycoengineered Pichia pastoris

A robust and scalable purification process was developed to quickly generate antibody of high purity and sufficient quantity from glycoengineered Pichia pastoris fermentation. Protein A affinity chromatography was used to capture the antibody from fermentation supernatant. A pH gradient elution was applied to the Protein A column to prevent antibody precipitation at low pH. Antibody from Protein A chromatography contained some product related impurities, which were the misassembling of cleaved heavy chain, heavy chain and light chain. It also had some process related impurities, including Protein A residues, endotoxin, host cell DNA and proteins. Cation exchange chromatography with optimal NaCl gradient at pH 4.5-6.0 efficiently removed these product and process related impurities. The antibody from glycoengineered P. pastoris was comparable to its commercial counterpart in heterotetramer folding, physical stability and binding affinity.     

High‐throughput process development of purification alternatives for the protein avidin

With an increased number of applications in the field of the avidin‐biotin technology, the resulting demand for highly‐purified protein avidin has drawn our attention to the purification process of avidin that naturally occurs in chicken egg white. The high‐throughput process development (HTPD) methodology was exploited, in order to evaluate purification process alternatives to commonly used ion‐exchange chromatography. In a high‐throughput format, process parameters for aqueous two‐phase extraction, selective precipitation with salts and polyethylene glycol, and hydrophobic interaction and mixed‐mode column chromatography experiments were performed. The HTPD strategy was complemented by a high‐throughput tandem high‐performance liquid chromatography assay for protein quantification. Suitable conditions for the separation of avidin from the major impurities ovalbumin, ovomucoid, ovotransferrin, and lysozyme were identified in the screening experiments. By combination of polyethylene glycol precipitation with subsequent resolubilization and separation in a polyethylene glycol/sulfate/sodium chloride two‐phase system an avidin purity of 77% was obtained with a yield >90% while at the same time achieving a significant reduction of the process volume. The two‐phase extraction and precipitation results were largely confirmed in larger scale with scale‐up factors of 230 and 133, respectively. Seamless processing of the avidin enriched bottom phase was found feasible by using mixed‐mode chromatography. By gradient elution a final avidin purity of at least 97% and yield >90% was obtained in the elution pool. The presented identification of a new and beneficial alternative for the purification of the high value protein thus represents a successful implementation of HTPD for an industrially relevant purification task. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:957–973, 2015