Production Of Monoclonal Antibodies Research Articles

Use of Chlamydial Elementary Bodies as Probes to Isolate Pathogen-Specific Human Monoclonal Antibodies.

Chlamydia trachomatis is one of the most prevalent sexually transmitted infectious agents in the world and the leading cause of infectious blindness. The role of antibodies in the prevention and clearance of infection is still not fully understood, but the analysis of the immunoglobulin response to novel vaccine candidates is an important part of many of these studies. In this chapter, we describe a novel method to identify and isolate Chlamydia-specific memory B cells by fluorescence-activated cell sorting (FACS) using fluorescently labeled whole bacteria from cryopreserved human PBMC samples. This method allows for live single cells to be sorted for cell culture, in vitro assays, single-cell RNA sequencing, and cloning of paired heavy and light chains for recombinant monoclonal antibody production.

Gemtuzumab ozogamicin in acute myeloid leukemia: past, present and future.

After being in the therapeutic wilderness for several decades, acute myeloid leukemia has been recently thrust into the limelight with a series of drug approvals. Technical refinements in production, genetic manipulation and chemical modification of monoclonal antibodies led to growing interest in antibodies-based treatment strategies. Much of the focus of these efforts in acute myeloid leukemia has been on CD33 as a target. On September 2, 2017, the U.S. Food and Drug Administration approved gemtuzumab ozogamicin for treatment of relapsed or refractory CD33+ acute myeloid leukemia. This signals a new chapter in the long and unusual story of gemtuzumab ozogamicin, which was the first antibody-drug conjugate approved for human use by the Food and Drug Administration. In this review we have analyzed the history of this drug which, among several mishaps, is experiencing a second youth and still represents a field to be further explored.

Portable Analytical Techniques for Monitoring Volatile Organic Chemicals in Biomanufacturing Processes: Recent Advances and Limitations.

It is essential to develop effective analytical techniques for accurate and continuous monitoring of various biomanufacturing processes, such as the production of monoclonal antibodies and vaccines, through sensitive and quantitative detection of characteristic aqueous or gaseous metabolites and other analytes in the cell culture media. A comprehensive summary toward the use of mainstream techniques for bioprocess monitoring is critically reviewed here, which illustrates the instrumental and procedural advances and limitations of several major analytical tools in biomanufacturing applications. Despite those drawbacks present in modern detection systems such as mass spectrometry, gas chromatography or chemical/biological sensors, a considerable number of useful solutions and inspirations such as electronic or optoelectronic noses can be offered to greatly overcome the restrictions and facilitate the development of advanced analytical techniques that can target a more diverse range of key nutritious components, products or potential contaminants in different biomanufacturing processes.

Advances in Production of Monoclonal Antibody and Antibody Fragments in Engineered Prokaryotes and Yeast

Advances in Production of Monoclonal Antibody and Antibody Fragments in Engineered Prokaryotes and Yeast

Generating and characterizing the anti-human CD45 monoclonal antibody

Introduction: CD45 is a common marker of leukocytes. Anti-human CD45 monoclonal antibody (MAb) has been used widely in diagnosing and monitoring hematologic diseases. The aim of this study was to generate an anti-human CD45 MAb, which can be used in research and diagnosis.
 Methods: Recombinant human CD45RO antigen was expressed from E. coli BL21 (DE3), purified and analyzed by SDS-PAGE and Western blotting. The purified CD45RO antigen was used to immunize Balb/c mice. Spleen cells from immunized mouse were collected and fused with P3X63Ag8.653 myeloma cells to form hybridoma. Anti-CD45 antibody-secreting capacity of hybridoma clones was evaluated by ELISA assay. Anti-CD45 MAb from the culture supernatant of the chosen hybridoma clone was purified by affinity chromatography. The MAb was characterized the biochemical characteristics and biological activity.
 Results: Recombinant human CD45RO antigen was expressed and purified from E.coli BL21 (DE3). Injection of purified CD45RO antigen provoked the immune response in Balb/c mice. Hybridoma clones were generated successfully by the fusion of spleen cells from the selected immunized-mouse and myeloma cells. Among these hybridoma clones, one with the highest yield of MAb production was identified. The isotype of the anti-CD45 MAb created in this work is IgG2b, while its the light chain is kappa (k) type. The affinity of this MAb with CD45RO antigen is high with Kd value at the picomolar level. The anti-CD45 MAb can interact with CD45 naturally expressed on the surface of Jurkat cells in Western blotting and fluorescent immuno-staining assay.
 Conclusion: We have developed successfully an anti-human CD45 MAb using hybridoma technology, which can recognize CD45 in ELISA, Western blotting, and fluorescent immuno-staining analysis. Although further investigations are necessary, obviously, our anti-human CD45 MAb is potential for research and diagnosis applications.

Effect of the disulfide isomerase PDIa4 on the antibody production of Chinese hamster ovary cells

Therapeutic monoclonal antibodies recognize and bind specific molecules on the surface of target cells, stimulating the immune system, which can attack these targeted cells. These antibodies are produced by mammalian cells, including Chinese hamster ovary (CHO) cells, because the formation of antibodies requires complicated posttranslational modifications, including peptidyl-prolyl cis/trans isomerization, disulfide bond formation, and glycosylation. Currently, it is thought that the efficient production of secretory proteins is limited by posttranslational processes. The ER is the biosynthesis site of all secreted and membrane proteins. The accumulation of unfolded proteins in the ER causes the ER stress response. During the ER stress state, various molecular chaperones are expressed to prevent proteins from the aggregate formation. The molecular chaperone involved in ER stress likely plays an essential role in the production of secretory proteins. The purpose of this study was to improve the production of monoclonal antibodies by cells. We elucidated the function of ER chaperones in the production of a monoclonal antibody. First, we quantitatively measured the mRNA expression levels of protein disulfide-isomerase family members. In CHO HcD6 cells treated with tunicamycin, the expression level of pdia4 was significantly increased. Second, we investigated the relationship between PDIa4 and antibody productivity in pdia4-knockdown cells. Both a decrease in the amount of secreted antibody and the accumulation of immature antibodies inside the cells were observed. Recombinant PDIa4 was able to refold the antibodies and Fabs. These results indicate that PDIa4 affects the production of monoclonal antibodies by catalyzing disulfide bond formation in these antibodies in CHO cells.

Kinetic analysis of effects of temperature and time on the regulation of venom expression in Bungarus multicinctus

Venom gland is a highly efficient venom production system to maintain their predatory arsenal. Venom toxins mRNA has been shown to increase abruptly in snake after venom expenditure, while the dynamics of venom accumulation during synthesis are poorly understood. Here, PacBio long-read sequencing, Illumina RNA sequencing (RNA-seq), and label-free proteome quantification were used to investigate the composition landscape and time- and temperature-dependent dynamics changes of the Bungarus multicinctus venom gland system. Transcriptome data (19.5223 Gb) from six adult B. multicinctus tissues were sequenced using PacBio RS II to generate a reference assembly, and average 7.28 Gb of clean RNA-seq data was obtained from venom glands by Illumina sequencing. Differentially expressed genes (DEGs) mainly were protein processing rather than venom toxins. Post-translational modifications provided the evidence of the significantly different proportions of toxins in the venom proteome with the changing of replenishment time and temperature, but constant of venom toxins mRNA in the venom gland transcriptome. Dynamic of toxins and genes involved in venom gland contraction suggesting the formation of the mature venom gland system would take at least 9 days. In addition, 59 toxin processing genes were identified, peptidylprolyl isomerase B of which underwent positive selection in Toxicofera. These results provide a reference for determining the extraction time of venom, production of polyclonal and monoclonal antibody for precise treatment plans of venomous snakebites, and construction of an in vitro synthesis system for snake venom protein.

Mechanistic insights into viral clearance during the chromatography steps in antibody processes by using virus surrogates

Viral safety is required for biological products to treat human diseases, and the burden of inactivation and or virus removal lies on the downstream purification process. Minute virus of mice (MVM) is a nonenveloped parvovirus commonly used as the worst-case model virus in validation studies because of its small size and high chemical stability. In this study, we investigated the use of MVM-mock virus particle (MVP) and bacteriophage ΦX174 as surrogates for MVM to mimic viral clearance studies, with a focus on chromatography operations. Based on structural models and comparison of log reduction value among MVM, MVP, and ΦX174, it was demonstrated that MVP can be used as a noninfectious surrogate to assess viral clearance during process development in multiple chromatography systems in a biosafety level one (BSL-1) laboratory. Protein A (ProA) chromatography was investigated to strategically assess the impact of the resin, impurities, and the monoclonal antibody product on virus removal.

FcRn Overexpression Expands Diversity of the Humoral Immune Response in bFcRn Transgenic Mice.

The neonatal Fc receptor (FcRn) plays key roles in IgG and albumin homeostasis, maternal IgG transport, and antigen presentation of IgG-opsonized antigens. Previously, we reported that transgenic (Tg) mice that overexpress bovine FcRn (bFcRn) have augmented T-dependent humoral immune response with increased IgG protection, higher level of antigen-specific antibodies, greater number of antigen-specific B cells, and effective immune response even against weakly immunogenic epitopes. In this study we analyzed the diversity of the humoral immune response of bFcRn Tg mice, using a length distribution analysis (spectratyping) and next generation sequencing (NGS) of the immunoglobulin heavy chain variable regions. Our analysis showed that in response to immunization with ovalbumin or transfected cells that expressed a unique membrane protein, our Tg animals developed a more diverse plasma cell repertoire than controls, which manifested in greater numbers of different clones, and clusters with fewer highly expanded large clones, as identified by the variable region (CDR3) of the immunoglobulin heavy chain. The increased antibody diversity in Tg mice after immunization was observed at both IgM and IgG levels, indicating that the increased humoral immune diversity in Tg mice is due to a higher number of both activated, antigen-specific naïve and isotype switched B cells. We thus demonstrated that the BCR repertoire of the immunized bFcRn Tg animals is more diverse compared to wild type mice, which likely makes these Tg mice a better choice for monoclonal antibody production against challenging antigens, including the extracellular regions of cell membrane proteins.

Expression of a recombinant anti-programed cell death 1 antibody in the mammary gland of transgenic mice

Nivolumab, a fully human IgG4 anti-programed cell death 1（PD-1）antibody, is recently one of the most popular and successful therapeutic monoclonal antibodies in clinical use. With the increasing demands for Nivolumab and other therapeutic monoclonal antibodies, the mammary gland bioreactor has been regarded as another choice for the production of recombinant monoclonal antibodies besides mammalian cell culture. Here, we expressed a recombinant human anti-PD-1 antibody in the mammary glands of transgenic mice. Two expression vectors were constructed bearing the heavy and light chains of anti-PD-1 antibody respectively under the control of bovine αs1-casein promoter. Transgenic mice were then generated by co-microinjection of the two expression cassettes. Three F0 founders with both heavy chain and light chain positive were obtained. Transgenes of both chains were detected to be stably transmitted to the offspring. The recombinant antibody was detected in the milk of transgenic mice with the highest expression level up to 80.52 ± 0.82 mg/L and could specifically binds to the human PD-1 antigen. Therefore, our results suggest the feasibility of anti-PD-1 antibody production in the milk of transgenic animals.

Simultaneous Monitoring and Comparison of Multiple Product Quality Attributes for Cell Culture Processes at Different Scales Using a LC/MS/MS Based Multi-Attribute Method

In response to the FDA's call for applying Quality by Design (QbD) to the manufacturing process, the biopharmaceutical industry has invested extensively into the monitoring and controlling of product quality attributes for bioprocesses. To assure the safety and efficacy of the drug product, defining critical quality attributes (CQA) and understanding their correlation with critical process parameters (CPP) becomes vitally important. In this work, a liquid chromatography-mass spectrometry based multi-attribute method (MAM) has been applied to the monitoring and trending of multiple CQAs of a monoclonal antibody product. To the best of our knowledge, this is the first demonstration of applying MAM to both a 3-liter development mini-bioreactor (3 L bioreactor) and a 2000-liter GMP single use bioreactor (2000L SUB). MAM was proven not only to be a great analytical tool for monitoring product quality attributes throughout the time course of the cell culture process, it could also provide critical product quality information in order to understand any potential process performance differences during scale-up and/or technology transfer. The successful monitoring and trending of the multiple CQAs throughout the 17-day cell culture process lays a solid foundation for possible real time in-process control and release of biotherapeutics using MAM in the future.

Quality-targeting dynamic optimization of monoclonal antibody production

Compliance with Quality by Design (QbD) constitutes a major challenge in biopharmaceuticals. Monoclonal antibodies (mAbs) represent a significant biopharmaceutical product class, typically produced in mammalian cell cultures. A key quality attribute for mAb production is glycosylation. We examine how process intensification affects glycosylation via dynamic optimization using different problem formulations. We maximize process performance with simultaneous control of product quality. For these, we utilize a mechanistic dynamic model for mAb production in mammalian cell cultures including glycosylation presented by Ehsani et al. in Computer Aided Chemical Engineering (2017). To achieve target glycan distribution in the final product, we incorporate constraints for the acceptable glycosylation ranges into the dynamic optimization problem. As a result, we derive optimal supplementation profiles of nutrients and/or nucleotide sugars. This work successfully illustrates an example of how model-based dynamic optimization can be employed for implementation of the QbD approach in biopharmaceutics.

PH and excipient profiles during formulation of highly concentrated biotherapeutics using bufferless media.

Several models have been developed to describe the shifts in pH and excipient concentrations seen during diafiltration of monoclonal antibody (mAb) products accounting for both Donnan equilibrium and electroneutrality constraints. However, these models have assumed that the mAb charge is either constant or only a function of pH, assumptions that will not be valid when formulating highly concentrated mAbs using bufferless or low-buffered media due to the change in local H+ concentration at the protein surface. The objective of this study was to incorporate the effects of both pH and ionic strength on the mAb charge, through the use of a charge regulation model based on the amino acid sequence of the mAb, into an appropriate mass balance model to describe the pH and excipient profiles during diafiltration. The model involves no adjustable parameters, with the protein charge evaluated directly from the protonation/deprotonation of the ionizable amino acids accounting for the electrostatic interactions between the charged mAb and the H+ ions. Model predictions are in excellent agreement with experimental data for the pH and ion concentrations during diafiltration of a mAb and fusion protein with different isoelectric points and different formulation conditions. Model simulations are then used to obtain fundamental insights into the factors controlling the diafiltration behavior as well as guidelines for development of diafiltration processes to achieve target bufferless formulation conditions.

Inter-laboratory study of an optimised peptide mapping workflow using automated trypsin digestion for monitoring monoclonal antibody product quality attributes

Peptide mapping analysis is a regulatory expectation to verify the primary structure of a recombinant product sequence and to monitor post-translational modifications (PTMs). Although proteolytic digestion has been used for decades, it remains a labour-intensive procedure that can be challenging to accurately reproduce. Here, we describe a fast and reproducible protocol for protease digestion that is automated using immobilised trypsin on magnetic beads, which has been incorporated into an optimised peptide mapping workflow to show method transferability across laboratories. The complete workflow has the potential for use within a multi-attribute method (MAM) approach in drug development, production and QC laboratories. The sample preparation workflow is simple, ideally suited to inexperienced operators and has been extensively studied to show global applicability and robustness for mAbs by performing sample digestion and LC-MS analysis at four independent sites in Europe. LC-MS/MS along with database searching was used to characterise the protein and determine relevant product quality attributes (PQAs) for further testing. A list of relevant critical quality attributes (CQAs) was then established by creating a peptide workbook containing the specific mass-to-charge (m/z) ratios of the modified and unmodified peptides of the selected CQAs, to be monitored in a subsequent test using LC-MS analysis. Data is provided that shows robust digestion efficiency and low levels of protocol induced PTMs.Graphical abstract

Implementing PAT for single-pass tangential flow ultrafiltration for continuous manufacturing of monoclonal antibodies

Control of single pass tangential flow ultrafiltration is crucial for continuous manufacturing of monoclonal antibodies. We propose a strategy to control the output concentration of a continuous ultrafiltration step regardless of variations in the volume or concentration of the feed material. Scheduling algorithms for the filtration runs and cleaning cycles are developed, accounting for the expected cycle times of different unit operations in the continuous train. The complete design space for the membrane module is characterized experimentally. An empirical model of the design space is developed to predict the logarithmic relationship between the feed flowrate and the maximum concentration factors achievable in a single pass for feed concentrations in the range of 1–50 g/L and feed flowrates in the range of 10–210 mL/min. The control strategy leverages in-line concentration, flowrate and pressure sensors, including near infrared spectroscopy (NIRS) flow cells to measure the concentration of mAb in the feed and retentate streams in the range 0.5–200 g/L. The control elements are the permeate pump and a variable control valve on the retentate line, which are used to obtain the desired concentration factor across the module in a single pass. Two case studies are designed to test the control system under conditions of high feed flow rate and low feed concentration, and low feed flow rate and high feed concentration, respectively. Successful control is demonstrated in both cases. The proposed system is the first complete approach that integrates process understanding, advanced monitoring sensors, and control strategies to consistently achieve concentration targets over long continuous campaigns for production of monoclonal antibodies.

A new detection technique for fluoroquinolone-conjugated proteins by high performance liquid chromatography with UV/fluorescence detectors

This study was aimed to develop a simple, fast, and reliable technique to detect fluoroquinolones (FQs) -conjugated bovine serum albumin (BSA). Four tested FQs, enrofloxacin, ofloxacin, danofloxacin, and orbifloxacin, were conjugated with BSA by following the N-hydroxysuccinimide ester method. The technique was designed according to the different absorption characteristics of the FQs and BSA; FQs can be detected by both UV and fluorescence detectors, but BSA can only be detected by UV. The results demonstrated that the developed method was efficient in detecting FQs-BSA conjugates. In addition, the method not only trace the FQs and BSA conjugation responses but also can be used to estimate the level of FQs-BSA conjugation. Therefore, this technique is a valuable tool for the detection drug-carrier-conjugated antigens, especially for FQs-BSA-conjugates during the production of anti-FQs monoclonal antibodies.

Comparative study for the production of monoclonal antibodies in single‐use vs stainless steel bioreactors based on product quality and stress factor

AbstractSingle‐use bioreactors (SUB) and stainless steel bioreactors (SSB) differ in their physical properties. This can have an effect on the performed processes. To be able to guarantee flexible planning in a facility, any kind of processes in SUB and SSB must be sufficiently similar. To date, assessments of SUB and SSB have been exclusively specific for one single product. This study shows a general nonproduct‐specific comparison of the production of different types of monoclonal antibodies in different scales of SUBs and SSBs. The study is set up with 72 clinical manufacturing fermentation runs in total by comparing the cell culture performance and quality attributes of these two bioreactor systems. The outcome is that SUBs and SSBs can produce monoclonal antibodies with comparable cell culture performance and product quality. This general comparability paves the way for SUBs to produce new and existing products for (clinical) manufacturing.

Production and characterisation of monoclonal antibodies against immunoglobulins of Cirrhinus mrigala (Hamilton 1822)

Serum immunoglobulins (Ig) of mrigal Cirrhinus mrigala (Hamilton 1822) immunised with bovine serum albumin (BSA), were purified by affinity chromatography using BSA-CL agarose column. The purified mrigal Ig (m-Ig) was characterised under reducing condition by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) which revealed two bands of 85 and 26 kDa corresponding to heavy and light chain, respectively. Following fusion of splenocytes from Balb/c mice immunised with purified m-Ig with myeloma cells, three hybridomas showing reactivity with m-Ig were cloned by limiting dilution. The monoclonal antibodies (MAbs) generated by these clones were designated as 3B2-E12, 3B2-F9 and 4C3-B2 and characterised by western blotting and isotyping. Western blot analysis of the supernatant from the three clones with purified m-Ig indicated that, all the three MAbs were specific to heavy chain. Isotyping revealed that 3B2-E12 MAb was of IgG1 isotype whereas the other two MAbs were of IgG2a isotype. Cross reactivity of anti-mrigal Ig MAb (3B2-E12) was observed with serum Ig of Catla catla and Labeo rohita indicating semi-conserved nature of Ig in Indian major carps.

Optimization of stereospecific targeting technique for selective production of monoclonal antibodies against native ephrin type-A receptor 2

High priority stereospecific targeting (SST) featuring selective production of conformation-specific monoclonal antibodies was directed against a native receptor, EphA2 (ephrin type-A receptor 2). A critical point for this technology is selection of sensitized B lymphocytes by antigen-expressing myeloma cells through their B-cell receptors (BCRs). The essential point is that antigens expressed on myeloma cells retain their original three dimensional structures and only these are recognized. Immunization with recombinant plasmid vectors as well as antigen-expressing CHO cells elicits enhanced sensitization of target B lymphocytes generating stereospecific antibodies. More than 24% of hybridoma-positive wells were identified to be cell-ELISA positive, confirming high efficiency. IgG-typed conformation-specific monoclonal antibodies could be also produced by the SST technique. Immunofluorescence analysis confirmed specific binding of sensitized B lymphocytes to antigen-expressing myeloma cells. Furthermore, stereospecific monoclonal antibodies to EphA2 specifically recognized EphA2-expressing cancer cells as demonstrated by Cell-ELISA. In the present study, we were able to develop priority technology for selective production of conformation-specific monoclonal antibodies against an intact receptor EphA2, known to be overexpressed by epithelial tumor cells of multiple cancer types.

Investigation into the impact of tyrosine on the product formation and quality attributes of mAbs in rCHO cell cultures.

Tyrosine (Tyr) is crucial to the maintenance of the monoclonal antibody (mAb) titers and quality attributes in fed-batch cultures of recombinant Chinese hamster ovary (rCHO) cells. However, the relation between tyrosine and these aspects is not yet fully defined. In order to further elucidate such a relation, two groups of fed-batch experiments with high tyrosine (H-T) or low tyrosine (L-T) additions producing an IgG1 monoclonal antibody against CD20 were implemented to investigate the intracellular and extracellular effects of tyrosine on the culture performance. It was found that the scarcity of tyrosine led to the distinctive reduction in both viable cell density and antibody specific production rate, hence the sharply reduced titer, possibly related to the impaired translation efficiency caused by the substrate limitation of tyrosine. In addition, alterations to the critical quality attributes were detected in the L-T group, compared to those in the H-T condition. Notable decrease in the contents of intact antibody was found under the L-T condition because of the elevated reductive level in the supernatant. Moreover, the aggregate content in the L-T condition was also reduced, probably resulting from the accumulation of extracellular cystine. In particular, the lysine variant content noticeably increased with tyrosine limitation owing to the downregulation of two carboxypeptidases, i.e., CpB and CpH. Overall, understanding the role of tyrosine in these aspects is fundamental to the increase of product titers and control of critical quality attributes in the monoclonal antibody production of rCHO cell fed-batch cultures. KEY POINTS: • Tyrosine is essential in the maintenance of product titers and the control of product qualities in high cell density cultivations in rCHO cell. • This study revealed the bottleneck of decreased qmAbupon the deficiency of tyrosine. • The impact of tyrosine on the critical product qualities and the underlying mechanisms were also thoroughly assessed.