Early-stage cell line screening is a vital step in developing biosimilars of therapeutic monoclonal antibodies (mAbs). While the quality of the manufactured antibodies is commonly assessed by ...charge-based separation methods employing UV absorbance detection, these methods lack the ability to identify resolved mAb variants. We evaluated the performance of microfluidic capillary electrophoresis coupled to mass spectrometry (MCE-MS) as a rapid tool for profiling mAb biosimilar candidates from clonal cell lines. A representative originator sample was used to develop the MCE-MS method. The addition of dimethylsulfoxide (DMSO) to the background electrolyte yielded up to 60-fold enhancement of the protein MS signal. The resulting electropherograms consistently provided resolution of mAb charge variants within 10 min. Deconvoluted mass spectra facilitated the identification of basic variants such as C-terminal lysine and proline amidation, while the acidic variants could be assigned to deamidated forms. The MCE-MS method also allowed the identification of 18 different glycoforms in biosimilar samples. To mimic early-stage cell line selection, samples from five clonal cell lines that all expressed the same biosimilar candidate mAb were compared to their originator mAb. Based on the similarity observed in charge variants and glycoform profiles acquired by MCE-MS, the most promising candidate could be selected. The MCE-MS method demonstrated good overall reproducibility, as confirmed by a transferability study involving two separate laboratories. This study highlights the efficacy of the MCE-MS method for rapid proteoform screening of clonal cell line samples, underscoring its potential significance as an analytical tool in biosimilar process development.
•Microfluidic CE-MS allows charge-variant and glycoform profiling of therapeutic mAbs.•The addition of DMSO to the BGE provides over 60 fold signal enhancement.•Repeated analyses and transferability study indicate good method reproducibility.•mAb charge variants and glycoforms were assigned based on the acquired CE-MS data.•Microfluidic CE-MS enables rapid and sensitive screening of biosimilar candidates.
From 2006 to 2011, an average of 15 novel recombinant protein therapeutics have been approved by US Food and Drug Administration (FDA) annually. In addition, the expiration of blockbuster biologics ...has also spurred the emergence of biosimilars. The increasing numbers of innovator biologic products and biosimilars have thus fuelled the demand of production cell lines with high productivity. Currently, mammalian cell line development technologies used by most biopharmaceutical companies are based on either the methotrexate (MTX) amplification technology or the glutamine synthetase (GS) system. With both systems, the cell clones obtained are highly heterogeneous, as a result of random genome integration by the gene of interest and the gene amplification process. Consequently, large numbers of cell clones have to be screened to identify rare stable high producer cell clones. As such, the cell line development process typically requires 6 to 12 months and is a time, capital and labour intensive process. This article reviews established advances in protein expression and clone screening which are the core technologies in mammalian cell line development. Advancements in these component technologies are vital to improve the speed and efficiency of generating robust and highly productive cell line for large scale production of protein therapeutics.
Asymmetric Immunoglobulin G (IgG)-like antibody is a fast-growing class of therapeutics among various bispecific antibody formats. However, the expression of correctly assembled products with high ...purity and yield remains challenging. Approaches have been investigated to facilitate antibody heterodimerization, including protein engineering, cell line development (CLD) screening, and vector element customization. In this paper, an innovative CLD platform was developed to express HLX101, an asymmetric IgG antibody consisting of two different heavy chains and two common light chains. We combined Leap-In Transposase® technology, a novel transposon-based integration strategy with two ORF “dual” and four ORF “quad” expression vector designs, and a microcapillary electrophoresis (µM) based high-throughput screening method, which efficiently identified clones expressing heterodimers at early stages of CLD. In this single case study of HLX101, the percentage of single clones expressing desired heterodimer among outgrown clones isolated from the stable pool was increased from 6% to 35% by varying dual vector ratios during co-transfection. This number was further improved to 90 % by a “quad” vector design. The final titer of a single clone fed-batch production reached 6 g/L. The established CLD platform demonstrated a simple and effective way to obtain high-yield and high-quality cell lines for asymmetric antibody production.
•A CLD platform was established to improve asymmetric antibody assembly and purity.•A stable pool with 90 % of clones expressing desired heterodimers was generated.•The single clone production titer of BsAbs reached high titer of 6 g/L.•Single vector design mitigated imbalanced expression levels of multiple chains.
The Golgi apparatus is a common and highly dynamic organelle in eukaryotic cells. It plays an important role in secretory trafficking and cargo modifications. Increasing evidence suggests that ...structural changes and functional disorders of the Golgi apparatus are involved in many human diseases, but whether Golgi dysfunction is a causal factor in regard to the progression of these diseases remains unknown. GM130 has been postulated to play roles in Golgi stack formation and vesicular transport based on studies on cultured cells and in vitro reconstitutions. To define the role of GM130 in animal, a GM130 knockout mouse has recently been created. Based on the principle of homologous recombination, the GM130 conditional knockout mouse model was established through gene targeting, stem cell screening, and blastocyst injection. Such model has been successfully applied for studies of physiological functions of GM130 and Golgi apparatus at the cellular and animal levels.
Objective To establish an array tagged high-throughput sequencing and pre-polyclonal screening strategy for fast identification of genome-edited cell clones. Methods Introducing of G to T point ...mutation at rs826415 site of K562 cells by CRISPR/Cas9-mediated homologous recombination. The candidate cells were then seeded by 10~20 cells per well in a 96-well-plateand and so to be expanded into 96 polyclones. A two-step nested PCR with array barcoded primers was used to amplify the rs826415 surrounding region to differentially tag PCR product from each polyclone. The PCR products were mixed and subjected to next-generation sequencing to detect the recombination and indel rate for each polyclone. The polyclonal cells carrying the highest ‘homology-directed repair (HDR) without indel’ rate were sorted for monoclonal growth and rs826415 genotyping. Results Polyclonal cells obtained by CRISPR/Cas9-mediated homologous recombination and limiting dilution were screened with array tagged high-throughput sequencing. Ratio
Mammalian cell line generation typically includes stable pool generation, single cell cloning and several rounds of clone selection based on cell growth, productivity and product quality criteria. ...Individual clone expansion and phenotype‐based ranking is performed initially for hundreds or thousands of mini‐scale cultures, representing the major operational challenge during cell line development. Automated cell culture and analytics systems have been developed to enable high complexity clone selection workflows; while ensuring traceability, safety, and quality of cell lines intended for biopharmaceutical applications. Here we show that comprehensive and quantitative assessment of cell growth, productivity, and product quality attributes are feasible at the 200–1,200 cell colony stage, within 14 days of the single cell cloning in static 96‐well plate culture. The early cell line characterization performed prior to the clone expansion in suspension culture can be used for a single‐step, direct selection of high quality clones. Such clones were comparable, both in terms of productivity and critical quality attributes (CQAs), to the top‐ranked clones identified using an established iterative clone screening approach. Using a complex, multi‐subunit antigen as a model protein, we observed stable CQA profiles independently of the cell culture format during the clonal expansion as well as in the batch and fed‐batch processes. In conclusion, we propose an accelerated clone selection approach that can be readily incorporated into various cell line development workstreams, leading to significant reduction of the project timelines and resource requirements.
In Pichia pastoris bioprocess engineering, classic approaches for clone selection and bioprocess optimization at small/micro scale using the promoter of the alcohol oxidase 1 gene (PAOX1), induced by ...methanol, present low reproducibility leading to high time and resource consumption.
An automated microfermentation platform (RoboLector) was successfully tested to overcome the chronic problems of clone selection and optimization of fed-batch strategies. Different clones from Mut+P. pastoris phenotype strains expressing heterologous Rhizopus oryzae lipase (ROL), including a subset also overexpressing the transcription factor HAC1, were tested to select the most promising clones.The RoboLector showed high performance for the selection and optimization of cultivation media with minimal cost and time. Syn6 medium was better than conventional YNB medium in terms of production of heterologous protein.The RoboLector microbioreactor was also tested for different fed-batch strategies with three clones producing different lipase levels. Two mixed substrates fed-batch strategies were evaluated. The first strategy was the enzymatic release of glucose from a soluble glucose polymer by a glucosidase, and methanol addition every 24 hours. The second strategy used glycerol as co-substrate jointly with methanol at two different feeding rates. The implementation of these simple fed-batch strategies increased the levels of lipolytic activity 80-fold compared to classical batch strategies used in clone selection. Thus, these strategies minimize the risk of errors in the clone selection and increase the detection level of the desired product.Finally, the performance of two fed-batch strategies was compared for lipase production between the RoboLector microbioreactor and 5 liter stirred tank bioreactor for three selected clones. In both scales, the same clone ranking was achieved.
The RoboLector showed excellent performance in clone selection of P. pastoris Mut+ phenotype. The use of fed-batch strategies using mixed substrate feeds resulted in increased biomass and lipolytic activity. The automated processing of fed-batch strategies by the RoboLector considerably facilitates the operation of fermentation processes, while reducing error-prone clone selection by increasing product titers.The scale-up from microbioreactor to lab scale stirred tank bioreactor showed an excellent correlation, validating the use of microbioreactor as a powerful tool for evaluating fed-batch operational strategies.
In high-throughput biotechnology and structural biology, molecular cloning is an essential prerequisite for attaining high yields of recombinant protein. However, a rapid, cost-effective, easy clone ...screening protocol is still required to identify colonies with desired insert along with a cross check method to certify the expression of the desired protein as the end product. We report an easy, fast, sensitive and cheap visual clone screening and protein expression cross check protocol employing gold nanoparticle based plasmonic detection phenomenon. This is a non-gel, non-PCR based visual detection technique, which can be used as simultaneous high throughput clone screening followed by the determination of expression of desired protein.
Infectious clone vectors used widely in genetic research. While constructing
soybean mosaic virus
(SMV) clone vectors, we found that transformed
Agrobacterium
grew significantly different depending ...on the viral strains used. In particular, the clone vectors constructed with SMV SC15 significantly suppressed the growth of
Agrobacterium
. Recombinant and truncated virus vector experiments showed that the polymorphism of a P1 protein coding sequence of SC15 leads to the growth inhibition of
Agrobacterium
. But the lack of other protein encoding sequences, except for the sequence encoding coat protein, should reduce the ability of SC15 to suppress
Agrobacterium
growth. A vector (pCB301-
att
L-SC15P) compatible with the Gateway cloning system was constructed using this
Agrobacterium
inhibitory sequence. The results from the LR recombination reaction with pCB301-
att
L-SC15P and
Agrobacterium
transformation showed the valuable application potential of the
Agrobacterium
inhibitory sequence to serve as a negative screening factor for effective recombinant clone screening in
Agrobacterium
.
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