Scale Production Of Monoclonal Antibodies Research Articles

Monoclonal Antibody Purification with Reduced Turbidity by using High-salt Elution Buffer During Protein a Chromatography

During purification of monoclonal antibodies, viral inactivation/removal step is a mandatory step to comply with the regulatory guidelines which commonly performs at low pH about 3.5 and further neutralizes it to a pH about 7.0. Neutralization of monoclonal antibodies results formation of high turbidity which is troublesome for further processing such as filtration and other chromatography processes and it eventually leads to filter clogging and increases column back pressure. In this work, we propose a new optimized process of affinity chromatography (Protein A) by using a high salt elution buffer which reduces turbidity more than 80%. A systematic study of different concentration of salts were analysed and it reveals that salt concentration greater than 100mM in Protein A elution buffer reduces turbidity during neutralization of monoclonal antibodies when compared to low salt concentration of elution buffer. We also found that elution of monoclonal antibodies during Protein A chromatography at pH 3.5 results higher reduction of turbidity compared to pH 3.0. These findings provide both cost and time effective for large scale production of monoclonal antibodies as it eliminates the use of multiple filters throughout the purification process.

Rational optimization of a monoclonal antibody for simultaneous improvements in its solution properties and biological activity.

Developability considerations should be integrated with lead engineering of antibody drug candidates in interest of their cost effective translations into medicines. To explore feasibility of this imperative, we have performed rational mutagenesis studies on a monoclonal antibody (MAB1) whose development was discontinued owing to manufacturability hurdles. Seven computationally designed variants of MAB1 containing single point (V44K, E59S, E59T and E59Y) and double (V44KE59S, V44KE59T and V44KE59Y) mutations in its light chain were produced in Chinese Hamster Ovary (CHO) cells and purified by using platform processes employed during commercial scale production of monoclonal antibodies. MAB1 and its variants were formulated in the same platform buffer and subjected to a battery of experiments to assess their solution behaviors, and biological activities. Five of the seven (71%) variants of MAB1 demonstrated improved biophysical attributes in multiple experimental testings. Contrary to the commonly expressed reservations about potential biological activity loss upon developability optimizations, the improvements in solution behavior of MAB1 also increased its biological activity up to ~180%. In particular, concentrate-ability and apparent solubility of V44KE59S improved to ~150% and ~160%, respectively. Its diffusion interaction parameter (kD) reduced to 28% and viscosity at ~100 mg/ml decreased to less than half of the corresponding values for MAB1. V44KE59S is also slightly more active and its transfections in CHO cells were more productive. It also degraded slower than MAB1 in three month long 25°C and 40°C formulation stability studies. These results open doors to an exciting realm of structure-based biologic drug design where developability and biological activity can be simultaneously optimized at the molecular engineering stages.

Development of monoclonal antibodies against axenic amastigotes of Leishmania infantum strain in Iran: implication for diagnosis of Kala-azar.

Objective(s):Leishmaniasis is endemic in 88 countries. Amastigote forms of Leishmania are experts at exploiting host cell processes to establish infection. Monoclonal antibodies are key reagents used in the diagnosis of infectious and non-infectious diseases. The aim of this study was to produce monoclonal antibodies against axenic amastigotes of the Leishmania infantum strain in Iran.Materials and Methods:First, standard strains were cultured and axenic amastigote antigens of L. infantum were obtained. Since then, BALB/c smice were immunized and antibody titers were determined. For hybridoma cell formation, lymphocytes isolated from spleen of immunized mice and myeloma cells were fused at a ratio of 10 to 1 in the presence of polyethylene glycol, followed by limiting dilution for the isolation of monoclones. Subsequently, antibody isotypes were determined by using the isotyping kit. The best clone was injected intraperitoneally to pristane-primed mice for large scale production of monoclonal antibodies. The specificity of antibody was determined with Western blotting.Results:Approximately 25 positive monoclones were obtained, of which four hybrids producing anti-amastigotes L. infantum monoclonal antibodies with high optical density (OD), selected and designated as 8D2 FVI6, 8D2 FVI3, 6G2 FV4 and 6G2 FV3. Results from isotype determination showed the IgG2b sub-class in 6G2FV2 and 8D2FVI6 monoclones.Conclusion:This study produced monoclonal antibody against amastigotes of Iranian strain of L. infantum for the first time. These antibodies have reactivity against Iranian strain of L. infantum and can be used in the diagnosis of Kala-azar.

Large Scale Generation and Characterization of Anti-Human CD34 Monoclonal Antibody in Ascetic Fluid of Balb/c Mice.

Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. 5 ml ascitic fluid was harvested from each mouse in two times. Evaluation of mAb titration was assessed by ELISA method. The ascitic fluid was examined for class and subclasses by ELISA mouse mAb isotyping Kit. mAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose. Purity of monoclonal antibody was monitored by SDS -PAGE and the purified monoclonal antibody was conjugated with FITC. Monoclonal antibodies with high specificity and sensitivity against human CD34 by hybridoma technology were prepared. The subclass of antibody was IgG1 and its light chain was kappa. The conjugated monoclonal antibody could be a useful tool for isolation, purification and characterization of human hematopoietic stem cells.

Anticorps monoclonaux thérapeutiques en cancérologie

Advances in bioengineering have lead to the possibility to conduct large scale production of monoclonal antibodies (MoAB) and to reduce progressively the murine component from 30% (chimeric MoAB) to 5% (humanized MoAB) to 0% (human MoAB). Three types of extracellular components are targeted in solid tumours : (1) Growth factors with transmembrane tyrosine kinase receptors either of tumour cells (IGF1) or endothelial cells (Bevacizumab). Bevacizumab has activity additive to that of chemotherapy in advanced colorectal, non squamous lung, ovarian, metastatic breast cancers and glioblastomas; (2) Extracellular domain of those transmembrane receptors : EGFR in colorectal cancer if no activating of KRAS with cetuximab and panitumumab, head and neck carcinomas with radiotherapy, and probably squamous lung cancers. Anti-ERBB2 MoAb are now a constitutive part of therapy of ERBB2 positive breast cancers at any stage; (3) Differenciation cluster regulating relationship ot tumour and stromal cells and in particular immunologic effectors. This is the case of anti-CTLA4 MoAB ipilimumab which activates and amplifies immunological cytotoxic response against melanoma with improved survival. These activities are achieved to the expense of class, target related toxicity conditioned by expression of the target on normal cells and or mechanism of action (immunological toxicity with ipilimumab). Of note a synergistic or additive activity with valid treatment regimens of targeted cancers and now targeted small molecules.

A comprehensive comparison of mixing, mass transfer, Chinese hamster ovary cell growth, and antibody production using Rushton turbine and marine impellers

Large scale production of monoclonal antibodies has been accomplished using bioreactors with different length to diameter ratios, and diverse impeller and sparger designs. The differences in these physical attributes often result in dissimilar mass transfer, mechanical stresses due to turbulence and mixing inside the bioreactor that may lead to disparities in cell growth and antibody production. A rational analysis of impeller design parameters on cell growth, protein expression levels and subsequent antibody production is needed to understand such differences. The purpose of this study was to examine the impact of Rushton turbine and marine impeller designs on Chinese hamster ovary (CHO) cell growth and metabolism, and antibody production and quality. Experiments to evaluate mass transfer and mixing characteristics were conducted to determine if the nutrient requirements of the culture would be met. The analysis of mixing times indicated significant differences between marine and Rushton turbine impellers at the same power input per unit volume of liquid (P/V). However, no significant differences were observed between the two impellers at constant P/V with respect to oxygen and carbon dioxide mass transfer properties. Experiments were conducted with CHO cells to determine the impact of different flow patterns arising from the use of different impellers on cell growth, metabolism and antibody production. The analysis of cell culture data did not indicate any significant differences in any of the measured or calculated variables between marine and Rushton turbine impellers. More importantly, this study was able to demonstrate that the quality of the antibody was not altered with a change in the impeller geometry.

Hydrophobic interaction chromatography in dual salt system increases protein binding capacity

Hydrophobic interaction chromatography (HIC) uses weakly hydrophobic resins and requires a salting-out salt to promote protein-resin interaction. The salting-out effects increase with protein and salt concentration. Dynamic binding capacity (DBC) is dependent on the binding constant, as well as on the flow characteristics during sample loading. DBC increases with the salt concentration but decreases with increasing flow rate. Dynamic and operational binding capacity have a major raw material cost/processing time impact on commercial scale production of monoclonal antibodies. In order to maximize DBC the highest salt concentration without causing precipitation is used. We report here a novel method to maintain protein solubility while increasing the DBC by using a combination of two salting-out salts (referred to as dual salt). In a series of experiments, we explored the dynamic capacity of a HIC resin (TosoBioscience Butyl 650M) with combinations of salts. Using a model antibody, we developed a system allowing us to increase the dynamic capacity up to twofold using the dual salt system over traditional, single salt system. We also investigated the application of this novel approach to several other proteins and salt combinations, and noted a similar protein solubility and DBC increase. The observed increase in DBC in the dual salt system was maintained at different linear flow rates and did not impact selectivity.

Maximizing productivity of chromatography steps for purification of monoclonal antibodies

The large scale production of monoclonal antibodies presents a challenge to design efficient and cost effective downstream purification processes. We explored a two stage resin screening approach to identify the best candidates to be utilized for the platform purification of monoclonal antibodies. The study focused on commercially available affinity resins including Protein A, mimetic and mixed-mode interaction resins as well as ion exchangers used in polishing steps. An initial screening using pure proteins was followed by a final screening where selected resins were utilized for the purification of MAbs in complex mixtures. Initial screenings aimed to measure the theoretical upper limit for dynamic binding capacity (DBC) at 1% breakthrough and productivity. We confirmed that DBC of affinity, mimetic and mixed-mode resins was a strong function of the linear velocity used for loading. Productivities >27 g/(L-h), were obtained for rProtein A FF, Mabselect and Prosep rA Ultra at 2 min residence time. For the cation exchangers, we identified UNOsphere S and Fractogel SO(3) as the best candidates for our purification based on DBC. For anion exchangers operated in flowthrough mode, Q Sepharose XL and UNOsphere Q were selected from the initial screening based on DBC and resolution of IgG from BSA. Finally, a three step purification scheme was implemented using the selected affinity and ion exchangers for the purification of IgG from complex feedstocks. We found that Mabselect followed by UNOsphere Q and UNOsphere S provided the best purification scheme for our applications based on productivity.

Large Scale Production and Characterization of Anti-Human IgG Monoclonal Antibody in Peritoneum of Balb/c MICE

Monoclonal antibodies are key reagents that are used in biomedical researches, diagnosis of immunodeficiency diseases such as IgG subclasses deficiency and treatment of diseases like infections and cancers. For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human IgG were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 mL Pristane. After 10 days, approximately 3 mL ascitic fluid was harvested from the peritoneum of each mouse. Ascitic fluid was assayed for the titer of monoclonal antibody in reaction with human IgG and its cross reactivity in reaction with IgM and IgA. The titer of mAb was 100,000 and didn’t show cross reactivity with IgM and IgA. Immunobloting was done for confirming the ELISA method. In immunobloting, only one sharp band in the heavy chain position of IgG was developed. The subclass of antibody was IgG1 and its light chain was kappa. Ascitic fluid was purified by ion exchange chromatography and the purified monoclonal antibody was conjugated with HRP. The conjugated monoclonal antibody could had application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of all other infectious diseases.

Evaluation of immobilized metal membrane affinity chromatography for purification of an immunoglobulin G 1 monoclonal antibody

The large scale production of monoclonal antibodies (McAbs) has gaining increased relevance with the development of the hybridoma cell culture in bioreactors creating a need for specific efficient bioseparation techniques. Conventional fixed bead affinity adsorption commonly applied for McAbs purification has the drawback of low flow rates and colmatage. We developed and evaluated a immobilized metal affinity chromatographies (IMAC) affinity membrane for the purification of anti-TNP IgG 1 mouse McAbs. We immobilized metal ions on a poly(ethylene vinyl alcohol) hollow fiber membrane (Me 2+-IDA-PEVA) and applied it for the purification of this McAbs from cell culture supernatant after precipitation with 50% saturation of ammonium sulphate. The purity of IgG 1 in the eluate fractions was high when eluted from Zn 2+ complex. The anti-TNP antibody could be eluted under conditions causing no loss of antigen binding capacity. The purification procedure can be considered as an alternative to the biospecific adsorbent commonly applied for mouse IgG 1 purification, the protein G-Sepharose.

Practical considerations in operation and scale-up of spin-filter based bioreactors for monoclonal antibody production.

Minimum spin-filter fouling and optimum cell retention at high specific perfusion rates are important for efficient operation of a spin-filter based continuous perfusion bioreactor. We examined the effect of operation conditions and spin-filter configuration on the performance of continuous perfusion bioreactors using a perfusion recycle scheme. This study showed that single cell suspensions foul a spin-filter screen, partially but irreversibly, in the early stages of the bioreactor run. A high perfusion rate and cell density contribute to screen fouling, while an increase in rotational velocity and screen surface area per reactor volume reduce screen fouling. An empirical model was developed to describe the effects of these parameters on the perfusion capacity of a spin-filter. Examination of screen samples by scanning electron microscopy confirmed that partial screen fouling occurs at relatively early stages of fermentation. Once the initial partial screen fouling has occurred, further fouling continues slowly due to cell growth on the screen surface, and this gradually leads to the overflow state. The rate of this gradual fouling phase probably depends upon the number of cells deposited on the screen surface during the initial partial fouling. The usefulness of this model was confirmed by successful scale-up of high-cell-density (> 10 x 10(6)/mL) long-term (> 30 days) continuous perfusion process for commercial scale production of monoclonal antibodies.

Laboratory scale production of monoclonal antibodies in a tumbling chamber

A simple device for laboratory scale production of monoclonal antibodies has been developed. Hybridomas were cultured in four individual dialysis tubes containing 40–50 ml medium with 10% foetal calf serum, surrounded by 1.5-2 litres supply medium without any serum supplement. Once placed on a roller the special design of the apparatus leads to an eccentric rotation, thus keeping the cells in a stable homogeneous suspension. The system is automatically gassed, and this makes long term cultivation possible. Several hybridomas were tested over a culture period of at least 3 weeks, with supply medium changes every 3–4 days. Cell densities of up to 2.5 × 10 7/ml and antibody concentrations of 0.3-1.9 mg/ml after purification were obtained. Results with this in vitro system allow a complete renunciation of the established in vivo method. The so called ‘tumbling chamber’ apparatus is easy to handle and to sterilize, is economic and universally adaptable in any research laboratory.

An oscillating bubble chamber for laboratory scale production of monoclonal antibodies as an alternative to ascitic tumours

A simple roller bottle was constructed to house three dialysis tubes, each with a capacity of 75 ml. Cells were grown inside the dialysis tubing, which was immersed in ordinary DMEM medium without serum supplement. Cultures of hybridomas at medium or low density (2 ×10 5 cells/ml) could be expanded directly in the dialysis tubes to attain a high cell density of the order of 10 7/ml. Continuous gentle stirring of the cells was possible, since the design causes a bubble to oscillate along the length of each tube. The six cell lines tested all gave antibody concentrations of between 1.1 and 2.3 mg/ml at 20 days. Such an in vitro apparatus obviates the need to employ ascites production, because it is as simple or simpler than the injection of mice, and the in vitro product is very rich in antibody, whilst containing low amounts of contaminating proteins.