Capture of human monoclonal antibodies from cell culture supernatant by ion exchange media exhibiting high charge density

A shortcut purification sequence for therapeutic proteins should consist of three steps: capture, purification, and polishing. Special emphasis has been put on direct capture of human monoclonal antibodies from culture supernatants with ion-exchangers avoiding pretreatment steps such as desalting, dilution, and other means to reduce the ionic strength. CM-HyperD, a cation-exchanger composed of an inorganic macroporous support filled with a viscoelastic gel with a high charge density was used. Capture of monoclonal antibodies from clarified hybridoma cell culture grown in media supplemented with fetal calf serum was investigated. Screening of different pH conditions and buffers for the load step showed that monoclonal antibodies were efficiently bound by CM-HyperD at pH 4.0 and 5.0 at an ionic strength equivalent to culture supernatant. Combination of negative purification with Q-Sepharose FF and capturing with CM-HyperD gave sufficient yield and resolution. Implementation of wash steps with higher conductivity did not improve the purity, but decreased the yield. Interestingly, high flow rates improved the purity. When antibodies were captured from serumfree culture supernatant the antibody could be eluted in a single peak with substantial reduction of contaminants. Capturing of antibodies by ion-exchange sorbents from culture supernatant is possible despite the high salt content.     

Antigens in culture supernatant of Mycobacterium tuberculosis: epitopes defined by monoclonal and human antibodies.

Antigens of Mycobacterium tuberculosis found in the supernatant of heat-treated cultures were characterized in order to explore whether antigens from this source could be used for the development of a serological test. Culture supernatants and sonicates of 12, 25 and 39 d cultures were analysed by SDS-PAGE. In culture supernatant, major protein bands of 65, 24, and 12 kDa were visible after Coomassie brilliant blue staining. Using murine monoclonal antibodies in Western blots, a pattern of protein bands distinct from that of the corresponding M. tuberculosis sonicates was found in all the culture supernatants. Gel permeation chromatography, in the presence of SDS, was used to separate the major protein bands in the culture supernatant. In ELISA, sera from 20 of 26 patients with tuberculosis reacted with fractions containing mainly 24 kDa or 12 kDa proteins, whereas none of the control sera reacted. In Western blots, each patient serum had its own characteristic banding pattern with culture supernatant, but all the sera from tuberculosis patients and control subjects reacted with protein bands of 65, 61, 58, 30 and 24 kDa. The 12 kDa protein was recognized only by sera from patients with tuberculosis in both Western blots and ELISA. This suggests that different kinds of epitopes on proteins of M. tuberculosis are detected by human antibodies in Western blots and ELISA. We assume that epitopes recognized in Western blots by patients with tuberculosis and control subjects are ubiquitous and are also present on normal commensal bacteria. Epitopes recognized by only some patients with tuberculosis in Western blots may be linear and M. tuberculosis specific. Epitopes recognized by tuberculosis patients but by none of the control subjects in ELISA may be conformation related and M. tuberculosis specific. The major protein bands found in supernatants of heat-treated cultures, 24 and 12 kDa, possess epitopes that may be M. tuberculosis specific and are potentially valuable for the development of a serological test.     

Continuous precipitation of process related impurities from clarified cell culture supernatant using a novel coiled flow inversion reactor (CFIR)

Coiled Flow Inverter Reactor (CFIR) has recently been explored for facilitating continuous operation of several unit operations involved in downstream processing of biopharmaceuticals such as viral inactivation and protein refolding. The application of CFIR for continuous precipitation of clarified cell culture supernatant has been explored. The pH based precipitation is optimized in the batch mode and then in the continuous mode in CFIR using a design of experiments (DOE) study. Improved clearance of host cell DNA (52× vs. 39× in batch), improved clearance of host cell proteins (HCP) (7× vs. 6× in batch) and comparable recovery (90 vs. 91.5 % in batch) are observed along with six times higher productivity. To further demonstrate wider applicability of CFIR in performing continuous precipitation, two more case studies involving use of two different precipitation protocols (CaCl₂ based and caprylic acid based) are also performed. In both cases, clearance of host cell DNA, HCP, and product recovery are found to be comparable or better in CFIR than in batch operations. Moreover, increase in productivity of 16 times (CaCl₂ based) and eight times (caprylic acid based) is obtained for the two precipitation protocols, respectively. The data clearly demonstrate that CFIR can be seamlessly integrated into a continuous bioprocess train for performing continuous precipitation of clarified cell culture supernatant. To our knowledge this is the first report of such use.     

Affinity chromatographic purification of human immunoglobulin a from chinese hamster ovary cell culture supernatant

Hexamer peptide ligand HWRGWV, initially screened from a solid phase combinatorial peptide library for immunoglobulins G (IgG) purification, is shown to also have potential for immunoglobulin A (IgA) purification. The determined dissociation constants for hIgA on HWRGWV resins at three different peptide densities from 0.11 to 0.55 meq/g fall in the range of 10^?6–10^?7 M, which are somewhat lower than those for hIgG. Although relatively low dynamic binding capacity (DBC) in the range of 9.2–16.8 mg IgA/mL resin at linear flow rates from 173 to 35 cm/h were obtained for IgA compared to IgG, the DBC value of HWRGWV for IgA is much greater than current commercially available affinity ligands. Although relatively lower binding affinity to secretory IgA compared to monomeric IgA was observed, the peptide ligand resins exhibit great potential for large‐scale purification of both human IgA and secretory IgA. Recoveries of 96.0% and 94.3%, and purities of 90.3% and 91.7% were achieved for human IgA and secretory IgA purification, respectively, from spiked Chinese hamster ovary cell culture supernatants without an extra afterwash step. Over 95% in purities were achieved for IgA and secretory IgA with an extra afterwash step; however, the recoveries would decrease at least 15% and 40% for IgA and secretory IgA, respectively. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Production of mouse monoclonal antibodies using a continuous cell culture fermenter and protein G affinity chromatography

The production of anti--fetoprotein monoclonal antibodies for diagnostic use was carried out in a stirred tank fermenter equipped with a double membrane stirrer for bubble free aeration and continuous medium perfusion. A serum-free medium supplemented with 4 mM L-glutamine and 2.0 g/l glucose with a protein content of only 780 g/ml was used for the production process. The harvested antibodies were concentrated 50-fold using a tangential ultrafiltration system and were then purified in a one step purification process by protein G affinity chromatography. The purity of the final product (90%) was controlled by SDS-polyacrylamide gel electrophoresis, gel exclusion chromatography and isoelectric focussing. For further quality controls of the product the immunoglobulin subclass and the isoelectric point were determined and the specificity of the purified mAb was tested by RIA using¹²⁵I labelled -fetoprotein.1.87 g of purified monoclonal antibodies were produced (90% purity) within 2 weeks. It was found that the use of this type of stirred tank fermenter combined with a one step purification process using protein G affinity chromatography represents a suitable method for the fast production of medium scale quantities (500 mg–5 g) of monoclonal antibodies for diagnostic use.Abbreviations AFP -Fetoprotein - BSA bovine serum albumine - FCS Fetal calf serum - HRP horseradish peroxidase - OPD o-phenylenediamine dihydrochloride - I.P. isoelectric point - IEF isoelectric focussing - PBS Phosphate buffered saline     

Affinity chromatographic purification of human immunoglobulin M from human B lymphocyte cell culture supernatant

Compared to immunoglobulin G purification with extensively studied affinity ligands such as protein A and protein G, little work has been done on affinity chromatographic purification of immunoglobulin M. Hexamer peptide ligand HWRGWV, previously shown to bind specifically to the Fc fragment of IgG, also demonstrated potential for IgM purification. This study presents further characterization and investigation of this ligand for its potential for purification of IgM. Different running conditions were employed in order to improve the recovery and purity of IgM. The final recovery and purity of the antibody is feedstock dependent, but can reach levels of both recovery and purity as high as 95%. The dependence of the recovery and purity on total loading amount and initial IgM concentration were investigated and discussed. Although relatively low dynamic binding capacities (DBC) in the range of 4.6–13.1 mg IgM/mL resin at linear flow rates from 173 to 35 cm/h were obtained for IgM compared to IgG because of the large molecular weight of IgM, the DBC value of HWRGWV for IgM is much greater than protein-based IgM affinity ligands found in the literature and is competitive with current commercially available affinity ligands, such as KAPTIVE-M, CaptureSelect IgM and Ultralink Immobilized Mannan Binding Protein.     

Isolation of a recombinant antibody from cell culture supernatant: continuous annular versus batch and expanded-bed chromatography

Annular chromatography represents a crossflow approach to chromatographic separations, that allows the continuous separation of multicomponent mixtures. The potential of the method for continuous bioseparation has been discussed for some time, however, we demonstrate for the first time the processing of a complex feed (cell culture supernatant) taken from an actual (bio)process. Moreover, while previously published applications of annular chromatography concentrated on noninteractive (gel filtration) or nonspecific (ion exchange) chromatography, we show the possibility of continuous annular affinity chromatography. In particular, a commercially available preparative continuous annular chromatography (P-CAC) system was used to purify a recombinant antibody (human IgG(1)-kappa) from CHO cell culture supernatants by (pseudo)affinity chromatography on hydroxyapatite (HA) and rProtein A. Methods developed using small (2 mL) batch columns could be directly transferred to the P-CAC, where they yielded similar results in terms of final product quality. Yields were between 87% and 92% in the case of HA and between 77% and 82% in the case of rProtein A chromatography. DNA removal was nearly quantitative in all cases. Concomitantly, the antibody fraction of the total protein content was raised by one order of magnitude in HA and by a factor of 50 by rProtein A chromatography. In addition, a novel HA material (particle diameter -120 microm) was investigated, which was compatible with expanded-bed applications. However, the final purity of the antibody thus obtained and also the yields (<70%) were less than satisfactory.     

Recovery of a monoclonal antibody from hybridoma culture supernatant by affinity precipitation with Eudragit S-100

An IgG₁ monoclonal antibody (MAB) was isolated from hybridoma culture supernatant by affinity precipitation with an Eudragit S-100-based heterobifunctional ligand. Affinity binding was performed in a homogeneous aqueous phase at pH 7.5 followed by precipitation of the bound affinity complex by lowering the pH to 4.8. After two washing steps, elution of specifically bound MAB was achieved by incubating the precipitate with 0.1 M glycine.HCl pH 2.5. The influence of elution volume and time on the recovery of active MAB and the overall purification factor were studied. The best conditions enabled the recovery of 50.2% of active MAB with a purification factor of 6.2. A further dialysis against 50 mM Tris.HCl pH 8.0 increased the activity yield and the purification factor to 68.4% and 8.3, respectively. This result showed that part of the antibody activity loss during affinity precipitation was due to a reversible inactivation process, being easily recovered after a refining dialysis step.     

Purification of recombinant human growth hormone from CHO cell culture supernatant by Gradiflow preparative electrophoresis technology

Purification of recombinant human growth hormone (rhGH) from Chinese hamster ovary (CHO) cell culture supernatant by Gradiflow large-scale electrophoresis is described. Production of rhGH in CHO cells is an alternative to production in Escherichia coli, with the advantage that rhGH is secreted into protein-free production media, facilitating a more simple purification and avoiding resolubilization of inclusion bodies and protein refolding. As an alternative to conventional chromatography, rhGH was purified in a one-step procedure using Gradiflow technology. Clarified culture supernatant containing rhGH was passed through a Gradiflow BF200 and separations were performed over 60 min using three different buffers of varying pH. Using a 50 mM Tris/Hepes buffer at pH 7.5 together with a 50 kDa separation membrane, rhGH was purified to approximately 98% purity with a yield of 90%. This study demonstrates the ability of Gradiflow preparative electrophoresis technology to purify rhGH from mammalian cell culture supernatant in a one-step process with high purity and yield. As the Gradiflow is directly scalable, this study also illustrates the potential for the inclusion of the Gradiflow into bioprocesses for the production of clinical grade rhGH and other therapeutic proteins.     

High‐throughput screening techniques for rapid PEG‐based precipitation of IgG4 mAb from clarified cell culture supernatant

Locating optimal protein precipitation conditions for complex biological feed materials is problematic. This article describes the application of a series of high‐throughput platforms for the rapid identification and selection of conditions for the precipitation of an IgG₄ monoclonal antibody (mAb) from a complex feedstock using only microliter quantities of material. The approach uses 96‐microwell filter plates combined with high‐throughput analytical methods and a method for well volume determination for product quantification. The low material, time and resource requirements facilitated the use of a full factorial Design of Experiments (DoE) for the rapid investigation into how critical parameters impact the IgG₄ precipitation. To aid the DoE, a set of preliminary range‐finding studies were conducted first. Data collected through this approach describing Polyethylene Glycol (PEG) precipitation of the IgG₄ as a function of mAb concentration, precipitant concentration, and pH are presented. Response surface diagrams were used to explore interactions between parameters and to inform selection of the most favorable conditions for maximum yield and purification. PEG concentrations required for maximum yield and purity were dependant on the IgG₄ concentration; however, concentrations of 14 to 20% w/v, pH 6.5, gave optimal levels of yield and purity. Application of the high‐throughput approach enabled 1,155 conditions to be examined with less than 1 g of material. The level of insights gained over such a short time frame is indicative of the power of microwell experimentation in allowing the rapid identification of appropriate processing conditions for key bioprocess operations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Continuous purification of antibodies from cell culture supernatant with aqueous two-phase systems: From concept to process

An aqueous two-phase extraction (ATPE) process based on a PEG/phosphate system was developed for the capture of human immunoglobulin G and successfully applied to a Chinese hamster ovary and a PER.C6® cell supernatant. A continuous ATPE process incorporating three different steps (extraction, back-extraction, and washing) was set up and validated in a pump mixer-settler battery. Most of the higher molecular weight cell supernatant impurities were removed during the extraction step, while most of the lower molecular weight impurities were removed during the subsequent steps. A global recovery yield of 80% and a final protein purity of more than 99% were obtained for the IgG purification from a CHO cell supernatant, representing a 155-fold reduction in the protein/IgG ratio. For the purification of IgG from a PER.C6® cell supernatant, a global recovery yield of 100%, and a host cell protein purity were attained, representing a 22-fold reduction in the host cell protein/IgG ratio. These results, thus, open promising perspectives for the application of the developed ATPE process as a platform for the capture of antibodies. In fact, this new process has shown the ability to successfully recover and purify different antibodies from distinct cell culture supernatants. This technology can also overcome some of the limitations encountered using the typical chromatographic processes, besides inherent advantages of scalability, process integration, capability of continuous operation, and economic feasibility.     

Macrophage suppression by a low-molecular-weight fraction of murine spleen cell culture supernatant

Macrophage suppression has been reported to be mediated by a component of murine serum. The present investigation involves in vitro production of this macrophage modulator (suppressor) by concanavalin A-stimulated murine spleen cells. Spleen cell culture supernatant containing this suppressor, which has been called macrophage suppressor factor (MSF), caused a significant decrease in in vitro phagocytosis of Listeria monocytogenes by resident murine peritoneal macrophages. The molecular weight of MSF was determined by ultrafiltration to be less than 10,000, and the suppressor activity of MSF was not altered by heating at 100 degrees C for 30 min or storage at -70 degrees C for 6 months. MSF is resistant to treatment with Pronase E, but is, however, sensitive to acid hydrolysis. Activity of MSF in spleen cell culture supernatants from normal mice does not differ from that in supernatants from mice immunized with L. monocytogenes. It was determined that MSF is not affected by antigenic stimulation and is apparently produced constitutively.     

Monitoring of the production of monoclonal antibodies by hybridomas. Part II: Characterization and purification of acid proteases present in cell culture supernatant.

An acid proteolytic activity has been found in cell culture supernatants from long-term cultivations of hybridoma cells in hollow fibre bioreactors using serum free medium. The proteolytic activity has now been further characterized and the main results were: (1) the proteolytic activity showed a maximum around pH 3 and declined essentially to zero at pH 8; (2) the activity was specifically inhibited by pepstatin A; (3) the acid proteases consisted of two sets of closely spaced bands with apparent molecular weights of 40-45K and 90-105K, respectively; (4) the protease bands (40-45K and 90-105K) were reactive with anti-human cathepsin D; (5) the IEP values of the acid proteases ranged from pH 4.55-6.5. Furthermore, IgG incubation with the acid proteases isolated from hybridoma cells yielded fragments similar to those found in serum-free hollow fibre cell culture supernatants. These results indicated that the IgG fragments are the result of degradation by cathepsin D like proteases released after cell death or cell lysis.     

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     

Killing of human tumor cell lines by human monocytes and murine monoclonal antibodies

We evaluated the capacity of freshly isolated blood monocytes to mediate antibody-dependent cellular-mediated cytotoxicity (ADCC) in cooperation with murine anti-tumor monoclonal antibodies (MAbs). Blood monocytes isolated from most donors by adherence selection to fibronectin-coated plastic surfaces and subsequently depleted of natural killer/killer (NK/K) cells exhibited significant ADCC activity against tumor cell lines in combination with an IgG3 antitumor MAb (BR55-2). However, significant variation in ADCC competence was observed among donors. Culture parameters influencing monocyte ADCC activity were evaluated and optimized. The influence of MAb isotype on ADCC capacity of anti-tumor MAbs was also evaluated using anti-tumor class-switch variant hybridoma proteins and a panel of anti-tumor MAbs. MAbs of the IgG2a and IgG3 subclasses exhibited high ADCC potential, whereas MAbs of the IgG2b subclass exhibited no ADCC activity. One of two IgG1 MAbs tested exhibited high ADCC activity with monocyte effectors. The role of monocytes or macrophages in tumor remission of cancer patients undergoing MAb immunotherapy is not known. However, correlative studies of monocyte ADCC capacity and responsiveness of cancer patients to MAb immunotherapy may help to establish the role of these effectors in MAb-mediated tumor remissions.     

Application of thiophilic membranes for the purification of monoclonal antibodies from cell culture media

The application of thiophilic membranes for the purification of monoclonal antibodies from hybridoma culture media was studied. Affinity filtrations were performed with membrane stacks and also in a cross flow module with a spiral filtration channel. Purification factors up to five and concentration factors of about eight could be achieved. The flux behaviour was analysed and interpreted according to existing models of filtration. The results were confirmed by scanning electron microscopy. The binding capacity of the membranes differed considerably with the mode of operation. The main component responsible for membrane fouling was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and amino acid sequence analysis as bovine serum albumin or its fragments.     

On-line monitoring of monoclonal antibodies in animal cell culture using a grating coupler

A grating coupler was used for the on-line determination of monoclonal antibodies produced in perfused animal cell bioreactor. The device was connected with the culture vessel via a flow-injection analysis (FIA) system, which was controlled automatically. Specific antimouse lgG antibodies were immobilized on the surface of the sensor-chip. After injection of the sample, the binding of mouse lgG was observed in real time. The regeneration of the binding sites of the immobilized antibodies using an acidic solution allowed the on-line detection of produced monoclonal antibodies in the range of 10 to 150 mug/mL. In contrast to other techniques coupled to bioprocesses, the developed method represents a regenerable direct immunosensor. Results were compared with standard ELISA techniques (off-line) and a competitive immunochemical assay using the grating coupler (off-line). (c) 1993 John Wiley & Sons, Inc.