MabSelect SuRe Research Articles

Clearance of the rodent retrovirus, XMuLV, by protein A chromatography.

Protein A chromatography is the most common unit operation used in the manufacture of therapeutic monoclonal antibodies (mAbs) due to its high affinity and specificity for the IgG Fc domain. However, protein A chromatography is often not effective for viral clearance. Typical log reduction values (LRV) for the model retrovirus XMuLV range between 1 and 4 logs, while effective steps such as viral filtration can achieve 5-7 logs of clearance. XMuLV LRVs obtained on protein A are reproducible for a given mAb, but can vary widely for different mAbs, even with the same operating conditions. In order to understand the mechanism of XMuLV clearance on protein A, we have investigated its partitioning on Mabselect SuRe protein A resin and explored how the virus interacts with resin, product, and impurities. The results show that XMuLV has some interaction with the resin backbone and ligand, but also appears to bind to and coelute with the mAb. The interaction with product was further examined by evaluating the effect of feed conditions, loading, and different washes on XMuLV partitioning on the column. Understanding the mechanism of XMuLV removal on a protein A, resin provides insight into the variability and low viral clearance of this step and suggests ways in which the removal of virus by this step can be improved.

Effect of cleaning agents and additives on Protein A ligand degradation and chromatography performance

Protein A chromatography, employing the recombinant Protein A ligand, is widely used as a capture step for antibody and Fc-fusion proteins manufacture. Protein A ligands in these matrices are susceptible to degradation/loss when exposed to cleaning agents such as sodium hydroxide, resulting in loss of capacity on reuse. In this study, MabSelect Protein A ligand and MabSelect SuRe Protein A ligand were chosen to evaluate the impact of alkaline cleaning solutions on the ligands and the packed columns. The Protein A ligands alone and the Protein A columns were incubated or cycled in different concentrations of sodium hydroxide solutions with and without additives, respectively. Ligand integrity (degradation) and ligand function (binding affinity) were studied using SDS–PAGE and customized Biacore technology, surface plasma resonance (SPR) and were successfully correlated with column performance measurement in terms of static binding capacity (SBC), dynamic binding capacity (DBC) and recovery as a function of exposure to cleaning agents with and without additives. The findings and the methodology presented in this study are not only able to determine appropriate cleaning conditions for Protein A chromatography, but also provided tools to enable systematic and rapid study of the cleaning solutions and conditions.

Preparation and high resolution analysis of a Fab fragment

In this work a Fab fragment was produced by papain cleavage of a purified monoclonal antibody (Mab) followed by purification on MabSelect™ Sure™ and Capto™ L media (protein A and protein L, respectively, immobilized onto high flow agarose). Fractions from the different steps were analyzed by gel filtration (GF) to follow purification progress. The process started with Mab being purified from feed on MabSelect Sure and thereafter the Mab was digested by papain overnight at 37 degrees into Fab‐ and Fcfragments. Cleavage was interrupted with the inhibitor antipain and the digest was applied to a column containing MabSelect Sure. Fc and partially digested Mab bind to the chromatography medium whereas the Fab fragment elutes in the flow through together with other contaminants such as papain. The flow through fraction was applied to Capto L medium and the Fab fragment was thus further purified. The purity of the Fab fragment was confirmed by analysis on new high resolution GF media. This new GF media has the resolution power to separate both Mab from aggregates and Fab fragments from Fc fragments.

Studying host cell protein interactions with monoclonal antibodies using high throughput protein A chromatography

Protein A chromatography is typically used as the initial capture step in the purification of monoclonal antibodies produced in Chinese hamster ovary (CHO) cells. Although exploiting an affinity interaction for purification, the level of host cell proteins in the protein A eluent varies significantly with different feedstocks. Using a batch binding chromatography method, we performed a controlled study to assess host cell protein clearance across both MabSelect Sure and Prosep vA resins. We individually spiked 21 purified antibodies into null cell culture fluid generated with a non-producing cell line, creating mock cell culture fluids for each antibody with an identical composition of host cell proteins and antibody concentration. We demonstrated that antibody-host cell protein interactions are primarily responsible for the variable levels of host cell proteins in the protein A eluent for both resins when antibody is present. Using the additives guanidine HCl and sodium chloride, we demonstrated that antibody-host cell protein interactions may be disrupted, reducing the level of host cell proteins present after purification on both resins. The reduction in the level of host cell proteins differed between antibodies suggesting that the interaction likely varies between individual antibodies but encompasses both an electrostatic and hydrophobic component.

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2. Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 . The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix. This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A tool for increasing the lifetime of chromatography resins

There is a steadily increasing demand for speed, cost efficiency, and process understanding within biopharmaceutical process development. To match this, a high-throughput method for screening of cleaning-in-place (CIP) conditions for chromatography resins has been developed. The methodology includes fouling of MabSelect SuRe chromatography resin in 96-well filter plates, cleaning of the fouled resin by incubation in different CIP agents, and finally, analysis of the residual impurities on the resin after cleaning. This article describes the improvements that transformed the method from low throughput and significant manual interference to a totally automated method with high throughput and good reproducibility.

Evaluation of new affinity chromatography resins for polyclonal, oligoclonal and monoclonal antibody pharmaceuticals

The performance of MabSelect SuRe and IgSelect affinity chromatography resins designed for process-scale purification of antibodies was investigated. Various antibodies (4 human monoclonal, 1 human polyclonal and 1 bovine polyclonal antibody and 1 Fc-fusion protein) were used to evaluate the elution pH and dynamic binding capacity of the resins. The elution pH for each human antibody was similar on MabSelect SuRe and IgSelect (pH 3.5–3.8). No significant differences in dynamic binding capacity were observed among human antibodies on MabSelect SuRe (∼20–40 mg/mL resin) and IgSelect (∼10–30 mg/mL resin). The binding capacity order for the human antibodies was the same on MabSelect SuRe and IgSelect. Using a linear pH gradient, both resins were able to partially separate monomeric and aggregated forms of the antibodies. The results indicate that these new affinity resins are powerful tools for the purification of human polyclonal antibodies from transgenic animals and oligoclonal antibodies from CHO cell cultures.

A small bispecific protein selected for orthogonal affinity purification

A novel protein domain with dual affinity has been created by randomization and selection. The small alkali-stabilized albumin-binding domain (ABD*), used as scaffold to construct the library, has affinity to human serum albumin (HSA) and is constituted of 46 amino acids of which 11 were randomized. To achieve a dual binder, the binding site of the inherent HSA affinity was untouched and the randomization was made on the opposite side of the molecule. Despite its small size and randomization of almost a quarter of its amino acids, a bifunctional molecule, ABDz1, with ability to bind to both HSA and the Z2 domain/protein A was successfully selected using phage display. Moreover, the newly selected variant showed improved affinity for HSA compared to the parental molecule. This novel protein domain has been characterized regarding secondary structure and affinity to the two different ligands. The possibility for affinity purification on two different matrices has been investigated using the two ligands, the HSA matrix and the protein A-based, MabSelect SuRe matrix, and the new protein domain was purified to homogeneity. Furthermore, gene fusions between the new domain and three different target proteins with different characteristics were made. To take advantage of both affinities, a purification strategy referred to as orthogonal affinity purification using two different matrices was created. Successful purification of all three versions was efficiently carried out using this strategy.

In this issue: Biotechnology Journal 6/2010

AbstractMagnetizable antibody‐like proteinsDehal et al., Biotechnol. J. 2010, 5, 596–605Paramagnetic particles can be used to isolate molecules or cells from complex media: particles are manufactured and coated with a biological molecule that confers specific biorecognition. Incubation with a sample plus exposure to magnetic fields isolates the species of interest. Researchers from Dundee (UK) have designed, produced and assessed magnetized fusion proteins consisting of the antigen‐binding portion of an antibody (single chain variable fraction; scFv) fused to the heavy chain of the iron‐binding protein ferritin. The fusion protein subunits expressed in E. coli assemble to form a flower‐like fusion protein consisting of a ferritin sphere with scFvs on the surface. These “organic” magnetizable particles are likely to overcome some of the problems of toxicity associated with the use of traditional inorganic micro‐ and nanoparticles. Orthogonal affinity purificationAlm et al., Biotechnol. J. 2010, 5, 605–617Researchers in the lab of Sophia Hober (Stockholm, Sweden) created a protein domain with dual affinity. They used randomization and selection on a small alkali‐stabilized albumin‐binding domain (ABD*) with affinity to human serum albumin (HSA). The resulting bifunctional molecule, ABDz1, has the ability to bind to both HSA and the Z2 domain/protein A. ABDz1 showed improved affinity for HSA and was purified to homogeneity on two different matrices: the HSA matrix and the protein A‐based, MabSelect SuRe matrix. Furthermore, gene fusions between the new domain and three different target proteins with different characteristics were made. To take advantage of both affinities, a purification strategy referred to as orthogonal affinity purification was created. Successful purification of all three fusion proteins was efficiently carried out using this strategy.Feed the stem cellsBrowning et al., Biotechnol. J. 2010, 5, 588–595Current challenges in embryonic stem cell (ESC) research include the inability of sustaining and culturing of undifferentiated ESCs over time. Growth‐arrested feeder cells are essential for culturing and sustaining them. These feeder cells are currently prepared using γ‐radiation and chemical inactivation which both have severe limitations. Researchers from Norfolk (VA, USA) developed a new, simple and effective technique based on pulsed electric fields (PEFs) to produce viable growth‐arrested cells (RTS34st). They used them as high‐quality feeder cells to culture and sustain undifferentiated zebrafish ESCs and found that the cellular effects of PEFs depended directly upon the duration, number and electric field strength of the pulses. Thus, one can tune these fields to produce various types of growth‐arrested cells for culturing undifferentiated ESCs.

Bacteriophage and impurity carryover and total organic carbon release during extended protein A chromatography

In the biopharmaceutical industry, column chromatography residuals are routinely assessed by the direct measurement of mock eluates. In this study, we evaluated virus and other impurity carryover between protein A cycles and the feasibility of using a total organic carbon (TOC) analyzer to monitor for column impurity leakage as a correlate for actual measured carryover in mock eluates. Commercial process intermediates were used in scaled down studies of two protein A media, ProSep A (Millipore, Bedford, MA, USA) and MabSelect SuRe (GE Healthcare, Uppsala, Sweden). The chromatography system was programmed to run up to 200 normal load/elution cycles with periodic blank cycles to measure protein and phage carryover, and water flush cycles to measure TOC release. Sustained phage carryover was evident in each study. Carryover and TOC release was lowest in the case where cleaning was most stringent (50 mM NaOH/0.5 M Na 2SO 4 with MabSelect SuRe). The TOC analysis at this time does not appear to be a viable practical means of measuring impurity carryover; direct measurements in mock eluates appears to be more predictive of column performance.

Robustness of virus removal by protein A chromatography is independent of media lifetime

The robustness of virus clearance with respect to protein A media reuse was demonstrated using media with four matrix chemistries: Protein A immobilized ProSep A, Poros A50, Protein A ceramic Hyper DF and MabSelect SuRe, an alkali resistant protein A ligand. Endogenous retrovirus clearance, step yield, impurity clearance and other performance parameters were evaluated periodically in media cycled up to 300 times. Media lifetime was generally limited by either declining step yield or media fouling. However, clearance of endogenous retrovirus remained in an acceptable range, either increasing or remaining constant. Multiply cycled media were tested for clearance of three viruses (SV40, X-MuLV, and MMV); clearance was comparable to naïve media. Overall, virus clearance by protein A chromatography appears to be extremely robust with respect to media age.

High‐Throughput Process Development: Determination of Dynamic Binding Capacity Using Microtiter Filter Plates Filled with Chromatography Resin

Steadily increasing demand for more efficient and more affordable biomolecule-based therapies put a significant burden on biopharma companies to reduce the cost of R&D activities associated with introduction of a new drug to the market. Reducing the time required to develop a purification process would be one option to address the high cost issue. The reduction in time can be accomplished if more efficient methods/tools are available for process development work, including high-throughput techniques. This paper addresses the transitions from traditional column-based process development to a modern high-throughput approach utilizing microtiter filter plates filled with a well-defined volume of chromatography resin. The approach is based on implementing the well-known batch uptake principle into microtiter plate geometry. Two variants of the proposed approach, allowing for either qualitative or quantitative estimation of dynamic binding capacity as a function of residence time, are described. Examples of quantitative estimation of dynamic binding capacities of human polyclonal IgG on MabSelect SuRe and of qualitative estimation of dynamic binding capacity of amyloglucosidase on a prototype of Capto DEAE weak ion exchanger are given. The proposed high-throughput method for determination of dynamic binding capacity significantly reduces time and sample consumption as compared to a traditional method utilizing packed chromatography columns without sacrificing the accuracy of data obtained.

Comparison of protein A affinity sorbents III. Life time study

Protein A affinity chromatography is a popular purification method for immunoglobulins applied at various scales, ranging from micro-tube up to 1000 l column format. Three novel high capacity protein A affinity chromatography media have been subjected to a lifetime study using 50 consecutive purification cycles of a cell culture supernatant (CCS) containing a monoclonal antibody. Chromatographic conditions followed protocols used in industrial antibody processing, including stripping and cleaning-in-place of the resins. For all three media, no significant loss of purification performance (measured by sodium dodecylsulfate polyacrylamide gel electrophoresis and analytical size-exclusion chromatography (SEC)) could be observed over 50 cycles. Eluate samples were analyzed for leaked protein A and host cell protein (HCP) content. MabSelect SuRe, the first protein A affinity medium compatible with alkaline regeneration conditions, exhibited the lowest leakage levels, in the range of 1–3 ppm. For the media MabSelect Xtra and ProSep-vA Ultra, leakage levels were in the range of 30–40 ppm. Host cell protein content of eluates from MabSelect Xtra and SuRe were between 300 and 700 ppm, whereas for ProSep-vA Ultra 3000–4000 ppm was achieved.

Comparison of protein A affinity sorbents: II. Mass transfer properties

Protein A affinity chromatography is the standard purification method for isolation of therapeutic antibodies. Due to improvements in expression technology and optimization of fermentation, culture supernatants with high antibody content must be processed. Recently protein A affinity media with improved adsorption characteristics have been developed. The agarose media MabSelect Xtra and MabSelect SuRe are recent developments of the existing protein A affinity medium MabSelect. MabSelect Xtra is designed to exhibit a higher binding capacity for IgG, and MabSelect SuRe is functionalized with an alkaline stabilized protein A. ProSep-vA Ultra is a porous glass medium with a pore size of 70 nm, also developed to improve the binding capacity. Adsorption was measured in a finite and infinite bath. Mass transfer in these systems could be well described by a model including film and pore diffusion. Mass transfer parameters were used to accurately predict IgG breakthrough in packed bed mode. The dynamic binding capacity of all three media did not change when residence time was at least 4 min. All three media are suited for capture of feed stocks with high antibody content.