Concurrent transfection of randomized transgene configurations into targeted integration CHO host is an advantageous and cost‐effective method for expression of complex molecules
Publication: Biotechnology Journal, December 1, 2020
Emily Dong1, Cynthia Lam1, Danming Tang1, Salina Louie1, Mandy Yim1, Ambrose J. William2,
William Sawyer3, Shirley Yip1, Joseph Carver1, Ali AlBarakat1, Joni Tsukuda1, Brad Snedecor1,
1 Cell Culture and Bioprocess Operations Department
2 Purification Development Department
3 Biochemical and Cellular Pharmacology Department
Genentech, Inc. 1 DNA Way, South San Francisco, California 94080
*Correspondence to: Shahram Misaghi
complex recombinant proteins are increasingly desired as potential therapeutic options for many disease indications and are commonly expressed in the mammalian Chinese hamster ovary (CHO) cells. Generally, stoichiometric expression and proper folding of all subunits of a complex recombinant protein are required to achieve the desired titers and product qualities for a complex molecule. Targeted integration (TI) cell line development (CLD), which entails the insertion of the desired transgene(s) into a predefined landing‐pad in the CHO genome, enables the generation of a homogeneous pool of cells from which clonally stable and high titer clones can be isolated with minimal screening efforts. Despite these advantages, using a single transgene(s) configuration with predetermined gene dosage might not be adequate for the expression of complex molecules. The goal of this study is to develop a method for seamless screening of many vector configurations in a single TI CLD attempt.
Main methods and major results
as testing vector configurations in transient expression systems is not predictive of protein expression in the stable cell lines and parallel TI CLDs with different transgene configurations is resource‐intensive, we tested the concept of randomized configuration targeted integration (RCTI) CLD approach for expression of complex molecules. RCTI allows simultaneous transfection of multiple vector configurations encoding, a complex molecule, to generate diverse TI clones each with a single transgene configuration but clone specific productivity and product qualities. Our findings further revealed a direct correlation between transgenes’ configuration/copy‐number and titer/product quality of the expressed proteins.
Conclusions and implications
RCTI CLD enabled, with significantly fewer resources, seamless isolation of clones with comparable titers and product quality attributes to that of several parallel standard TI CLDs. Therefore, RCTI introduces randomness to the TI CLD platform while maintaining all the advantages, such as clone stability and reduced sequence variant levels, that the TI system has to offer.
Semiautomated Small-Scale Purification Method for High-Throughput Expression Analysis of Recombinant Proteins
Publication: Methods in Molecular Biology 2019;2025:51-68
1 Department of Biomolecular Resources, Genentech, Inc., South San Francisco, CA, USA.
2 Department of Early Stage Cell Culture, Genentech, Inc., South San Francisco, CA, USA.
3 23andMe Inc., South San Francisco, CA, USA.
4 Department of Biomolecular Resources, Genentech, Inc., South San Francisco, CA, USA.
Abstract: The expression analysis of recombinant proteins is a challenging step in any high-throughput protein production pipeline. Often multiple expression systems and a variety of expression construct designs are considered for the production of a protein of interest. There is a strong need to triage constructs rapidly and systematically. This chapter describes a semiautomated method for the simultaneous purification and characterization of proteins expressed from multiple samples of expression cultures from the E. coli, baculovirus expression vector system, and mammalian transient expression systems. This method assists in the selection of the most promising expression construct(s) or the most favorable expression condition(s) to move forward into large-scale protein production.
High-Throughput Protein Production and Purification - Methods and Protocols
About this book
This book compiles key protocols instrumental to the study of high-throughput protein production and purification which have been refined and simplified over the years and are now ready to be transferred to any laboratory. Beginning with a section covering general procedures for high-throughput protein production, the volume continues with high-throughput protocols adapted to the production of specific protein families, as well as an extensive section on protocols combining high-throughput protein production and their micro-characterization. Written for the highly successful Methods in Molecular Biology series, chapters in this book include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Perfusion cell culture for the production of conjugated recombinant fusion proteins reduces clipping and quality heterogeneity compared to batch-mode processes.
Publication: J Biotechnol. 2019 Aug 20;302:26-31
doi: 10.1016/j.jbiotec.2019.06.006. Epub 2019 Jun 15.
Salivary N-glycosyalation as a biomarker of Oral Cancer: A pilot study
Publication: Glycobiology, cwz046, 27 June 2019
Proteins obtained from whole saliva were subjected to PNGase F enzymatic digestion. The resulting N-glycans were analysed with weak anion exchange chromatography, exoglycosidase digestions coupled to ultra-high performance liquid chromatography (UHPLC) and/or mass spectrometry (MS). To determine N-glycan changes, 23 individuals with or without cancerous oral lesions were analysed using HILIC-UPLC and peak based area relative quantitation was performed.
An abundant and complex salivary N-glycomic profile was identified. The main structures present in saliva were neutral oligosaccharides consisting of high mannose, hybrid and complex structures, followed by smaller fractions of mono and di-sialylated structures. To determine if differential N-glycosylation patterns distinguish between oral cancer and control groups, Mann-Whitney testing and Principle Component Analysis (PCA) were used. 11 peaks were shown to be statistically significant (p ≤ 0.05) while PCA analysis showed segregation of the two groups based on their glycan profile. N-glycosylation changes are active in the oral carcinogenic process, and may serve as biomarkers for early detection to reduce morbidity and mortality. Identifying which N-glycans contribute most in the carcinogenic process, may lead to their use in the detection, prognosis and treatment of OC.
Shedding Light into the Subcutis: A Mass Spectrometry Based Immunocapture Assay Enabling Full Characterization of Therapeutic Antibodies after Injection in Vivo
Publication: Anal Chem. 2019 Aug 6;91(15):9490-9499
DNA-Encoded Macrocyclic Peptide Library
Publication: Methods Mol Biol. 2019;2001:273-284
Direct imaging of the recruitment and phosphorylation of S6K1 in the mTORC1 pathway in living cells
Publication: Scientific Reports 9, Article number: 3408 (04 March 2019)
Impact of cell culture media additives on IgG glycosylation produced in CHO cells
Publication: Biotechnology & Bioengineering. 2018 Dec 15
Quantitative assessment of affinity selection performance using DNA-encoded chemical libraries
Publication: Chembiochem. 2018 Dec 13
Development of a Selection Method for Discovering Irreversible (Covalent) Binders from a DNA-Encoded Library
Publication: SLAS Discovery 2018 Nov 1:2472555218808454
Automated high throughput microscale antibody purification workflows for accelerating antibody discovery
Publication: MAbs. 2018 May-Jun; 10(4): 624–635.
To rapidly find “best-in-class” antibody therapeutics, it has become essential to develop high throughput (HTP) processes that allow rapid
assessment of antibodies for functional and molecular properties. Consequently, it is critical to have access to sufficient amounts of high quality
antibody, to carry out accurate and quantitative characterization. We have developed automated workflows using liquid handling systems to
conduct affinity-based purification either in batch or tip column mode. Here, we demonstrate the capability to purify >2000 antibodies per day
from microscale (1 mL) cultures. Our optimized, automated process for human IgG1 purification using MabSelect SuRe resin achieves ∼70%
recovery over a wide range of antibody loads, up to 500 µg. This HTP process works well for hybridoma-derived antibodies that can be purified by
MabSelect SuRe resin. For rat IgG2a, which is often encountered in hybridoma cultures and is challenging to purify via an HTP process, we
established automated purification with GammaBind Plus resin. Using these HTP purification processes, we can efficiently recover sufficient
amounts of antibodies from mammalian transient or hybridoma cultures with quality comparable to conventional column purification.
Pyruvate Kinase Muscle‐1 Expression Appears to Drive Lactogenic Behavior in CHO Cell Lines, Triggering Lower Viability and Productivity: A Case Study
CHO cell lines are used to express a variety of therapeutic proteins. However, lactogenic behavior displayed by some CHO cell lines during manufacturing processes may result in a decline in viability, productivity, and possible alterations in product quality. In cultured cells, lactate is produced during glycolysis through irreversible conversion of phosphoenolpyruvate to pyruvate and then lactate via sequential function of pyruvate kinase and lactate dehydrogenase (LDH) enzymes. In the process of cell line development (CLD), we identified two lactogenic cell lines expressing different antibody molecules. The lactogenic behaviors of these cell lines could be differentially mitigated through optimization of either nutrient feeds or culture pH, depending on the cell line. Analysis of various proteins involved in the glycolysis pathway revealed a direct correlation between the pyruvate kinase muscle-1 (PKM-1) isoform levels- and lactogenic behavior. CRISPR mediated knockout of the PKM-1 isoform abolished lactate accumulation even under lactogenic conditions. Furthermore, we identified a cell line lacking expression of both PKM-1 and PKM-2 enzymes capable of maintaining productivity, viability, and growth without displaying lactogenic behavior. Targeted deletion of PKM in CHO cells may be tolerated due to expression of PKL (liver) and PKR (red blood cell) isoforms of pyruvate kinase. All together, our findings suggest that PKM-1 up-regulation during antibody production could trigger lactogenic behavior and that this enzyme is dispensable for CHO cell survival.
Critical considerations for immunocapture enrichment LC-MS bioanalysis of protein therapeutics and biomarkers
Publication: Bioanalysis. 2018 Jul 1;10(13):987-995. Epub 2018 Jul 4.
In recent years, immunocapture enrichment coupled with LC-MS technology has seen more applications for the measurement of low abundant protein therapeutics and biomarkers in biological matrices. In this article, several critical considerations for the application of immunocapture enrichment to LC-MS bioanalysis of protein therapeutics and biomarkers, including reagent selection, reagent characterization, designing of capture format, etc. are discussed. All these considerations are critical in developing reliable and robust bioanalytical assays with high assay specificity and sensitivity. Successful examples using the immunocapture LC-MS approach in the quantification of biotherapeutic and low abundant protein biomarkers will also be discussed.
Development of a shake tube-based scale-down model for perfusion cultures
Publication: Biotechnol Bioeng. 2018 Jul 24
Conservation of oncofetal antigens on human embryonic stem cells enables discovery of monoclonal antibodies against cancer
Publication: Sci Rep. 2018 Aug 2;8(1):11608.
Monoclonal antibodies (mAbs) are used as targeted therapies against cancers. These mAbs kill cancer cells via various
mechanisms of actions. In this study, human embryonic stem cells (hESCs) was used as the immunogen to generate a panel of
antibodies. From this panel of mAbs, A19 was found to bind both hESC and various cancer cell lines. The antigen target of A19 was
identified as Erbb-2 and glycan analysis showed that A19 binds to a N-glycan epitope on the antigen. A19 was elucidated to
internalize into cancer cells following binding to Erbb-2 and hence developed as an antibody-drug conjugate (ADC). Using ADC as
the mechanism of action, A19 was able to kill cancer cells in vitro and delayed the onset of tumour formation in mice xenograft
model. When compared to Herceptin, A19 binds to different isoforms of Erbb-2 and does not compete with Herceptin for the
same epitope. Hence, A19 has the potential to be developed as an alternative targeted therapeutic agent for cancers expressing
Improved performance in mammalian cell perfusion cultures by growth inhibition
Publication: Biotechnology Journal
Mammalian cell perfusion cultures represent a promising alternative for the production of various biopharmaceuticals to the current fed‐batch technology. Long‐term operation at a fixed viable cell density (VCD) requires a viable culture and a constant removal of excessive cells. Product loss in the cell removing bleed stream deteriorates the process yield. In this study, we investigated the use of chemical and environmental growth inhibition on culture performance by either adding valeric acid (VA) to the production media or by reducing the culture temperature (33.0o C) with respect to control conditions (36.5o C, no VA). Low temperature significantly reduced cellular growth, thus resulting in lower bleed rates accompanied by a reduced product loss of 11% compared to 26% under control conditions. Additionally, the cell specific productivity of the target protein improved and maintained stable, thus leading to media savings per mass of product. VA showed initially an inhibitory effect on cellular growth. However, cells seemed to adapt to the presence of the inhibitor leading to a recovery of the cellular growth. Cell cycle and Western blot analyses supported the observed results. This work underlines the role of temperature as a key operating variable for the optimization of perfusion cultures.
Structure-Guided Combinatorial Engineering Facilitates Affinity and Specificity Optimization of Anti-CD81 Antibodies
Publication: Journal of Molecular Biology
doi: 10.1016/ j.jmb.2018.05.018
Hepatitis C viral infection is the major cause of chronic hepatitis that affects as many as 71 million people worldwide. Rather than target the rapidly shifting viruses and their numerous serotypes, four independent antibodies were made to target the host antigen CD81 and were shown to block Hepatitis C viral entry. The single-chain variable fragment of each antibody was crystallized in complex with the CD81 large extracellular loop (LEL) in order to guide affinity maturation of two distinct antibodies by phage display. Affinity maturation of antibodies using phage display has proven to be critical to therapeutic antibody development and typically involves modification of the paratope for increased affinity, improved specificity, enhanced stability or a combination of these traits. One antibody was engineered for increased affinity for human CD81 LEL that equated to increased efficacy while the second antibody was engineered for cross-reactivity with cynomolgus CD81 to facilitate animal model testing. The use of structures to guide affinity maturation library design demonstrates the utility of combining structural analysis with phage display technologies.
Multi-step high-throughput conjugation platform for the development of antibody-drug conjugates
Publication: Journal of Biotechnology
Antibody drug conjugates (ADCs) form a rapidly growing class of biopharmaceuticals which attracts a lot of attention throughout the industry due to its high potential for cancer therapy. They combine the specificity of a monoclonal antibody (mAb) and the cell-killing capacity of highly cytotoxic small molecule drugs. Site-specific conjugation approaches involve a multi-step process for covalent linkage of antibody and drug via a linker. Despite the range of parameters that have to be investigated, high-throughput methods are scarcely used so far in ADC development.
In this work an automated high-throughput platform for a site-specific multi-step conjugation process on a liquid-handling station is presented by use of a model conjugation system. A high-throughput solid-phase buffer exchange was successfully incorporated for reagent removal by utilization of a batch cation exchange step. To ensure accurate screening of conjugation parameters, an intermediate UV/Vis-based concentration determination was established including feedback to the process. For conjugate characterization, a high-throughput compatible reversed-phase chromatography method with a runtime of 7 min and no sample preparation was developed. Two case studies illustrate the efficient use for mapping the operating space of a conjugation process. Due to the degree of automation and parallelization, the platform is capable of significantly reducing process development efforts and material demands and shorten development timelines for antibody-drug conjugates.
Design and Application of a DNA-Encoded Macrocyclic Peptide Library
Publication: ACS Chem Biol. 2018 Jan 19;13(1):53-59
A DNA-encoded macrocyclic peptide library was designed and synthesized with 2.4 × 1012 members composed of 4-20 natural and non-natural amino acids. Affinity-based selection was performed against two therapeutic targets, VHL and RSV N protein. On the basis of selection data, some peptides were selected for resynthesis without a DNA tag, and their activity was confirmed.
Controlling the Glycosylation Profile in mAbs Using Time-Dependent Media Supplementation
Publication: Antibodies 2018 Volume 7, Issue 1
In order to meet desired drug product quality targets, the glycosylation profile of biotherapeutics such as monoclonal antibodies (mAbs) must be maintained consistently during manufacturing. Achieving consistent glycan distribution profiles requires identifying factors that influence glycosylation, and manipulating them appropriately via well-designed control strategies. Now, the cell culture media supplement, MnCl2, is known to alter the glycosylation profile in mAbs generally, but its effect, particularly when introduced at different stages during cell growth, has yet to be investigated and quantified. In this study, we evaluate the effect of time-dependent addition of MnCl2 on the glycan profile quantitatively, using factorial design experiments. Our results show that MnCl2 addition during the lag and exponential phases affects the glycan profile significantly more than stationary phase supplementation does. Also, using a novel computational technique, we identify various combinations of glycan species that are affected by this dynamic media supplementation scheme, and quantify the effects mathematically. Our experiments demonstrate the importance of taking into consideration the time of addition of these trace supplements, not just their concentrations, and our computational analysis provides insight into what supplements to add, when, and how much, in order to induce desired changes.
Agonists and Antagonists of Protease-Activated Receptor 2 Discovered within a DNA-Encoded Chemical Library Using Mutational Stabilization of the Target.
The discovery of ligands via affinity-mediated selection of DNA-encoded chemical libraries is driven by the quality and concentration of the protein target. G-protein-coupled receptors (GPCRs) and other membrane-bound targets can be difficult to isolate in their functional state and at high concentrations, and therefore have been challenging for affinity-mediated selection. Here, we report a successful selection campaign against protease-activated receptor 2 (PAR2). Using a thermo-stabilized mutant of PAR2, we conducted affinity selection using our >100-billion-compound DNA-encoded library. We observed a number of putative ligands enriched upon selection, and subsequent cellular profiling revealed these ligands to comprise both agonists and antagonists. The agonist series shared structural similarity with known agonists. The antagonists were shown to bind in a novel allosteric binding site on the PAR2 protein. This report serves to demonstrate that cell-free affinity selection against GPCRs can be achieved with mutant stabilized protein targets.
Randomness in DNA Encoded Library Selection Data Can Be Modeled for More Reliable Enrichment Calculation.
DNA Encoded Libraries (DELs) use unique DNA sequences to tag each chemical warhead within a library mixture to enable deconvolution following affinity selection against a target protein. With next-generation sequencing, millions to billions of sequences can be read and counted to report binding events. This unprecedented capability has enabled researchers to synthesize and analyze numerically large chemical libraries. Despite the common perception that each library member undergoes a miniaturized affinity assay, selections with higher complexity libraries often produce results that are difficult to rank order. In this study, we aimed to understand the robustness of DEL selection by examining the sequencing readouts of warheads and chemotype families among a large number of experimentally repeated selections. The results revealed that (1) the output of DEL selection is intrinsically noisy but can be reliably modeled by the Poisson distribution, and (2) Poisson noise is the dominating noise at low copy counts and can be estimated even from a single experiment. We also discuss the shortcomings of data analyses based on directly using copy counts and their linear transformations, and propose a framework that incorporates proper normalization and confidence interval calculation to help researchers better understand DEL data.
Use of a Protein Engineering Strategy to Overcome Limitations in the Production of ‘Difficult to Express’ Recombinant Proteins
Publication: Biotechnology and Bioengineering, Oct 2017
Certain recombinant proteins are deemed “difficult to express” in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of “difficult” recombinant targets. Biotechnol. Bioeng. 2017;114: 2348-2359.
Auto-induction of Pichia pastoris AOX1 promoter for membrane protein expression
Publication: Protein Expression and Purification, June 2017
Pichia pastoris is a highly successful recombinant protein expression system due to its ability to quickly generate large quantities of recombinant proteins in simple media. P. pastoris has been used to successfully generate milligram quantities of many important human membrane proteins, including G-protein coupled receptors, ion channels, and transporters, which are becoming increasingly important therapeutic targets. Despite these successes, protein expression in P. pastoris is still cumbersome due to a need to change growth media from glycerol media to methanol induction media, which minimizes inhibition of the AOX1 promoter by residual glycerol. Taking advantage of this behavior of the AOX1 promoter, we developed Buffered extra-YNB Glycerol Methanol (BYGM) auto-induction media (100 mM potassium phosphate pH 6.0, 2.68% w/v YNB, 0.4% v/v glycerol, 0.5% v/v methanol, and 8 × 10-5% w/v biotin) which not only simplified the proteinexpression process, but also optimized protein expression levels in P. pastoris. We successfully used this auto-induction method to overexpress the target in both MutS and Mut+ strains. Moreover, we show that this method can facilitate screening high-expressing clones, as well as enable parallel protein production in P. pastoris.
Streamlining Workflow and Automation to Accelerate Laboratory Scale Protein Production
Publication: Protein Expression and Purification, Vol 133, 160-169, May 2017
Protein production facilities are often required to produce diverse arrays of proteins for demanding methodologies including crystallography, NMR, ITC and other reagent intensive techniques. It is common for these teams to find themselves a bottleneck in the pipeline of ambitious projects. This pressure to deliver has resulted in the evolution of many novel methods to increase capacity and throughput at all stages in the pipeline for generation of recombinant proteins. This review aims to describe current and emerging options to accelerate the success of protein production in Escherichia coli. We emphasize technologies that have been evaluated and implemented in our laboratory, including innovative molecular biology and expression vectors, small-scale expression screening strategies and the automation of parallel and multidimensional chromatography.
The Tetrameric Plant Lectin BanLec Neutralizes HIV through Bidentate Binding to Specific Viral Glycans
Modulation and Modeling of Monoclonal Antibody N-linked Glycosylation in Mammalian Cell Perfusion Reactors
Parallel Experimental Design and Multivariate Analysis Provides Efficient Screening of Cell Culture Media Supplements to Improve Biosimilar Product Quality
Publication: Biotechnology and Bioengineering, February 2017
Rational and high-throughput optimization of mammalian cell culture media has a great potential to modulate recombinant protein product quality. We present a process design method based on parallel design-of-experiment (DoE) of CHO fed-batch cultures in 96-deepwell plates to modulate monoclonal antibody (mAb) glycosylation using medium supplements. To reduce the risk of losing valuable information in an intricate joint screening, 17 compounds were separated into five different groups, considering their mode of biological action. The concentration ranges of the medium supplements were defined according to information encountered in the literature and in-house experience. The screening experiments produced wide glycosylation pattern ranges. Multivariate analysis including principal component analysis and decision trees was used to select the best performing glycosylation modulators. Subsequent D-optimal quadratic design with four factors (three promising compounds and temperature shift) in shake tubes confirmed the outcome of the selection process and provided a solid basis for sequential process development at a larger scale. The glycosylation profile with respect to the specifications for biosimilarity was greatly improved in shake tube experiments: 75% of the conditions were equally close or closer to the specifications for biosimilarity than the best 25% in 96-deepwell plates. Biotechnol. Bioeng. 2017;114: 1448-1458.
Characterization of H type 1 and type 1 N-acetyllactosamine glycan epitopes on ovarian cancer specifically recognized by the anti-glycan monoclonal antibody mAb-A4
Publication: The Journal of Biological Chemistry, February 2017
Cancer-specific glycans of ovarian cancer are promising epitopes for targeting with monoclonal antibodies (mAb). Despite their potential, structural characterization of these glycan epitopes remains a significant challenge in mAb preclinical development. Our group generated the monoclonal antibody mAb-A4 against human embryonic stem cells (hESC), which also bound specifically to N-glycans present on 11 of 19 ovarian cancer (OC) and 8 of 14 breast cancer cell lines tested. Normal cell lines and tissue were unstained by mAb-A4. To characterize the N-linked glycan epitopes on OC cell lines targeted by mAb-A4, we used glycosidases, glycan microarray, siRNA, and advanced high sensitivity matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The mAb-A4 epitopes were found to be Fucα1-2Galβ1-3GlcNAcβ (H type 1) and Galβ1-3GlcNAcβ (type 1 LacNAc). These structures were found to be present on multiple proteins from hESC and OC. Importantly, endo-β-galactosidase coupled with MALDI-MS allowed these two epitopes, for the first time, to be directly identified on the polylactosamines of N-glycans of SKOV3, IGROV1, OV90, and OVCA433. Furthermore, siRNA knockdown of B3GALT5 expression in SKOV3 demonstrated that mAb-A4 binding was dependent on B3GALT5, providing orthogonal evidence of the epitopes’ structures. The recognition of oncofetal H type 1 and type 1 LacNAc on OC by mAb-A4 is a novel and promising way to target OC and supports the theory that cancer can acquire stem-like phenotypes. We propose that the orthogonal framework used in this work could be the basis for advancing anti-glycan mAb characterization.
Capillary electrophoresis analysis of N-glycosylation changes of serum paraproteins in multiple myeloma
Publication: Electrophoresis, January 2017
Multiple myeloma (MM) is an immedicable malignancy of the human plasma cells producing abnormal antibodies (also referred to as paraproteins) leading to kidney problems and hyperviscosity syndrome. In this paper, we report on the N-glycosylation analysis of paraproteins from total human serum as well as the fragment crystallizable region (Fc ) and fragment antigen binding (Fab ) κ/λ light chain fractions of papain digested immunoglobulins from multiple myeloma patients. CE-LIF detection was used for the analysis of the N-glycans after endoglycosidase (PNGase F) mediated sugar release and fluorophore labeling (APTS). While characteristic N-glycosylation pattern differences were found between normal control and untreated, treated and remission stage multiple myeloma patient samples at the global serum level, less distinctive changes were observed at the immunoglobulin level. Principal component analysis adequately differentiated the four groups (control and three patient groups) on the basis of total serum N-glycosylation analysis. 12 N-glycan features showed statistically significant differences (p <0.05) among various stages of the disease in comparison to the control at the serum level, while only six features were identified with similar significance at the immunoglobulin level, including the analysis of the partitioned Fc fragment as well as the Fab κ and Fab λ chains.
Excess reactive oxygen species production mediates monoclonal antibody-induced human embryonic stem cell death via oncosis
Publication: Cell Death and Differentiation, January 2017
Antibody-mediated cell killing has significantly facilitated the elimination of undesired cells in therapeutic applications. Besides the well-known Fc-dependent mechanisms, pathways of antibody-induced apoptosis were also extensively studied. However, with fewer studies reporting the ability of antibodies to evoke an alternative form of programmed cell death, oncosis, the molecular mechanism of antibody-mediated oncosis remains underinvestigated. In this study, a monoclonal antibody (mAb), TAG-A1 (A1), was generated to selectively kill residual undifferentiated human embryonic stem cells (hESC) so as to prevent teratoma formation upon transplantation of hESC-derived products. We revealed that A1 induces hESC death via oncosis. Aided with high-resolution scanning electron microscopy (SEM), we uncovered nanoscale morphological changes in A1-induced hESC oncosis, as well as A1 distribution on hESC surface. A1 induces hESC oncosis via binding-initiated signaling cascade, most likely by ligating receptors on surface microvilli. The ability to evoke excess reactive oxygen species (ROS) production via the Nox2 isoform of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is critical in the cell death pathway. Excess ROS production occurs downstream of microvilli degradation and homotypic adhesion, but upstream of actin reorganization, plasma membrane damage and mitochondrial membrane permeabilization. To our knowledge, this is the first mechanistic model of mAb-induced oncosis on hESC revealing a previously unrecognized role for NAPDH oxidase-derived ROS in mediating oncotic hESC death. These findings in the cell death pathway may potentially be exploited to improve the efficiency of A1 in eliminating undifferentiated hESC and to provide insights into the study of other mAb-induced cell death.
Discovery of a potent BTK inhibitor with a novel binding mode using parallel selections with a DNA-encoded chemical library
Publication: ChemBioChem, January 2017
We have identified and characterized novel potent inhibitors of Bruton’s tyrosine kinase (BTK) from a single DNA-encoded library of over 110 million compounds by using multiple parallel selection conditions, including variation in target concentration and addition of known binders to provide competition information. Distinct binding profiles were observed by comparing enrichments of library building block combinations under these conditions; one enriched only at high concentrations of BTK and was competitive with ATP, and another enriched at both high and low concentrations of BTK and was not competitive with ATP. A compound representing the latter profile showed low nanomolar potency in biochemical and cellular BTK assays. Results from kinetic mechanism of action studies were consistent with the selection profiles. Analysis of the co-crystal structure of the most potent compound demonstrated a novel binding mode that revealed a new pocket in BTK. Our results demonstrate that profile-based selection strategies using DNA-encoded libraries form the basis of a new methodology to rapidly identify small molecule inhibitors with novel binding modes to clinically relevant targets.
Quantitative Glycomics: A Combined Analytical and Bioinformatics Approach
Publication: Methods in Enzymology, 2017
Glycosylation is one of the most common and essential protein modifications. Glycans conjugated to biomolecules modulate the function of such molecules through both direct recognition of glycan structures and indirect mechanisms that involve the control of protein turnover rates, stability, and conformation. The biological attributes of glycans in numerous biological processes and implications in a number of diseases highlight the necessity for comprehensive characterization of protein glycosylation. This chapter reviews cutting-edge methods and tools developed to facilitate quantitative glycomics. This chapter highlights the different methods employed for the release and purification of glycans from biological samples. The most effective labeling methods developed for sensitive quantitative glycomics are also described and discussed. The chromatographic approaches that have been used effectively in glycomics are also highlighted.
CP40 from Corynebacterium pseudotuberculosis is an endo-β-N-acetylglucosaminidase
Publication: BMC Microbiology, Vol 16, 261, December 2016
C. pseudotuberculosis is an important animal pathogen that causes substantial economical loss in sheep and goat farming. Zoonotic infections in humans are rare, but when they occur they are often severe and difficult to treat. One of the most studied proteins from this bacterium, the secreted protein CP40 is being developed as a promising vaccine candidate and has been characterized as a serine protease. In this study we have investigated if CP40 is an endoglycosidase rather than a protease.
CP40 does not show any protease activity and contains an EndoS-like family 18 of glycoside hydrolase (chitinase) motif. It hydrolyzes biantennary glycans on both human and ovine IgGs. CP40 is not a general chitinase and cannot hydrolyze bisecting GlcNAc.
Taken together we present solid evidence for re-annotating CP40 as an EndoS-like endoglycosidase. Redefining the activity of this enzyme will facilitate subsequent studies that could give further insight into immune evasion mechanisms underlying corynebacterial infections in animals and humans.
Twoplex 12/13 C 6 aniline stable isotope and linkage specific sialic acid labelling 2D-LC-MS workflow for quantitative N –glycomics
Discovery of cofactor-specific, bactericidal Mycobacterium tuberculosis InhA inhibitors using DNA-encoded library technology
Publication: Proceedings of the National Academy of Sciences of the United States of America, November 2016
Millions of individuals are infected with and die from tuberculosis (TB) each year, and multidrug-resistant (MDR) strains of TB are increasingly prevalent. As such, there is an urgent need to identify novel drugs to treat TB infections. Current frontline therapies include the drug isoniazid, which inhibits the essential NADH-dependent enoyl-acyl-carrier protein (ACP) reductase, InhA. To inhibit InhA, isoniazid must be activated by the catalase-peroxidase KatG. Isoniazid resistance is linked primarily to mutations in the katG gene. Discovery of InhA inhibitors that do not require KatG activation is crucial to combat MDR TB. Multiple discovery efforts have been made against InhA in recent years. Until recently, despite achieving high potency against the enzyme, these efforts have been thwarted by lack of cellular activity. We describe here the use of DNA-encoded X-Chem (DEX) screening, combined with selection of appropriate physical properties, to identify multiple classes of InhA inhibitors with cell-based activity. The utilization of DEX screening allowed the interrogation of very large compound libraries (1011 unique small molecules) against multiple forms of the InhA enzyme in a multiplexed format. Comparison of the enriched library members across various screening conditions allowed the identification of cofactor-specific inhibitors of InhA that do not require activation by KatG, many of which had bactericidal activity in cell-based assays.
Robust factor selection in early cell culture process development for the production of a biosimilar monoclonal antibody
Publication: Biotechnology Progress, September 2016
This work presents a multivariate methodology combining principal component analysis, the Mahalanobis distance and decision trees for the selection of process factors and their levels in early process development of generic molecules. It is applied to a high throughput study testing more than 200 conditions for the production of a biosimilar monoclonal antibody at microliter scale. The methodology provides the most important selection criteria for the process design in order to improve product quality towards the quality attributes of the originator molecule. Robustness of the selections is ensured by cross-validation of each analysis step. The concluded selections are then successfully validated with an external data set. Finally, the results are compared to those obtained with a widely used software revealing similarities and clear advantages of the presented methodology. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:181-191, 2017.
Process performance and product quality in an integrated continuous antibody production process
Publication: Biotechnology and Bioengineering, August 2016
Continuous manufacturing is currently being seriously considered in the biopharmaceutical industry as the possible new paradigm for producing therapeutic proteins, due to production cost and product quality related benefits. In this study, a monoclonal antibody producing CHO cell line was cultured in perfusion mode and connected to a continuous affinity capture step. The reliable and stable integration of the two systems was enabled by suitable control loops, regulating the continuous volumetric flow and adapting the operating conditions of the capture process. For the latter, an at-line HPLC measurement of the harvest concentration subsequent to the bioreactor was combined with a mechanistic model of the capture chromatographic unit. Thereby, optimal buffer consumption and productivity throughout the process was realized while always maintaining a yield above the target value of 99%. Stable operation was achieved at three consecutive viable cell density set points (20, 60, and 40 × 106 cells/mL), together with consistent product quality in terms of aggregates, fragments, charge isoforms, and N-linked glycosylation. In addition, different values for these product quality attributes such as N-linked glycosylation, charge variants, and aggregate content were measured at the different steady states. As expected, the amount of released DNA and HCP was significantly reduced by the capture step for all considered upstream operating conditions. This study is exemplary for the potential of enhancing product quality control and modulation by integrated continuous manufacturing. Biotechnol. Bioeng. 2017;114: 298-307.
Identifying a Robust Design Space for Glycosylation During Monoclonal Antibody Production
Publication: Biotechnology Progress, June 2016
Glycan distribution has been identified as a “critical quality attribute” for many biopharmaceutical products, including monoclonal antibodies. Consequently, determining quantitatively how process variables affect glycan distribution is important during process development to control antibody glycosylation. In this work, we assess the effect of six bioreactor process variables on the glycan distribution of an IgG1 produced in CHO cells. Our analysis established that glucose and glutamine media concentration, temperature, pH, agitation rate, and dissolved oxygen (DO) had small but significant effects on the relative percentage of various glycans. In addition, we assessed glycosylation enzyme transcript levels and intracellular sugar nucleotide concentrations within the CHO cells to provide a biological explanation for the observed effects on glycan distributions. From these results we identified a robust operating region, or design space, in which the IgG1 could be produced with a consistent glycan distribution. Since our results indicate that perturbations to bioreactor process variables will cause only small (even if significant) changes to the relative percentage of various glycans (<±1.5%)-changes that are too small to affect the bioactivity and efficacy of this IgG1 significantly-it follows that the glycan distribution obtained will be consistent even with relatively large variations in bioreactor process variables. However, for therapeutic proteins where bioactivity and efficacy are affected by small changes to the relative percentage of glycans, the same analysis would identify the manipulated variables capable of changing glycan distribution, and hence can be used to implement a glycosylation control strategy. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1149-1162, 2016.
EndoSd: an IgG glycan hydrolyzing enzyme in Streptococcus dysgalactiae subspecies dysgalactiae
Publication: Future Microbiology, May 2016
AIM: The aim of this study was to identify and characterize EndoS-like enzymes in Streptococcus dysgalactiae subspecies dysgalactiae (SDSD).
MATERIALS & METHODS: PCR, DNA sequencing, recombinant protein expression, lectin blot, ultra high performance liquid chromatography analysis and a chitinase assay were used to identify ndoS-like genes and characterize EndoSd.
RESULTS: EndoSd were found in four SDSD strains. EndoSd hydrolyzes the chitobiose core of the glycan on IgG. The amino acid sequence of EndoSd is 70% identical to EndoS in S. pyogenes, but it has a unique C-terminal sequence. EndoSd secretion is influenced by the carbohydrate composition of the growth medium.
CONCLUSION: Our findings indicate that IgG glycan hydrolyzing activity is present in SDSD, and that the activity can be attributed to the here identified enzyme EndoSd.
High-throughput analysis of immunoglobulin G glycosylation
Publication: Expert Review of Proteomics, Vol 13, No 5, 523-534, May 2016
INTRODUCTION: Glycosylation of immunoglobulin G (IgG) is important for its effector functions and was shown to be related to age, sex and disease status of an individual. Adding glycomic information to genome-wide association studies (GWAS) and large clinical trials is enabling insight into the functional relevance of changes in glycosylation, as well as molecular mechanisms behind these changes. Large-scale studies require sensitive, robust and affordable high-throughput methodologies for glycosylation analysis, which are currently available in only a limited number of laboratories.
AREAS COVERED: This review focuses on currently used high-throughput approaches for N-glycosylation analysis of IgG, as well as some recent advances in the areas of deglycosylation, trypsin digestion, labeling, purification, derivatization and automation of current workflows. Relevant literature was searched using the PubMed database. Expert commentary: Development, optimization and validation of robust, affordable and simple high-throughput glycosylation analysis methods is essential for discovery and validation of diagnostic and prognostic glycan biomarkers. Although significant advances in glycosylation analysis have been made in recent years, currently used protocols will have to be further optimized to enable subsequent analysis of glycosylation on all levels with the limited initial sample and in the minimal amount of time, which is still a challenging task.
Downstream process development strategies for effective bioprocesses: Trends, progress, and combinatorial approaches
Publication: Engineering in Life Sciences, April 2016
The biopharmaceutical industry is at a turning point moving toward a more customized and patient-oriented medicine (precision medicine). Straightforward routines such as the antibody platform process are extended to production processes for a new portfolio of molecules. As a consequence, individual and tailored productions require generic approaches for a fast and dedicated purification process development. In this article, different effective strategies in biopharmaceutical purification process development are reviewed that can analogously be used for the new generation of antibodies. Conventional approaches based on heuristics and high-throughput process development are discussed and compared to modern technologies such as multivariate calibration and mechanistic modeling tools. Such approaches constitute a good foundation for fast and effective process development for new products and processes, but their full potential becomes obvious in a correlated combination. Thus, different combinatorial approaches are presented, which might become future directions in the biopharmaceutical industry.
Rapid N-glycan release from glycoproteins using immobilized PNGase F microcolumns
Publication: Journal of Chromatography. B, Analytical technologies in the Biomedical and Life Sciences, February 2016
N-glycosylation profiling of glycoprotein biotherapeutics is an essential step in each phase of product development in the biopharmaceutical industry. For example, during clone selection, hundreds of clones should be analyzed quickly from limited amounts of samples. On the other hand, identification of disease related glycosylation alterations can serve as early indicators (glycobiomarkers) for various pathological conditions in the biomedical field. Therefore, there is a growing demand for rapid and easy to automate sample preparation methods for N-glycosylation analysis. In this paper, we report on the design and implementation of immobilized recombinant glutathione-S-transferase (GST) tagged PNGase F enzyme microcolumns for rapid and efficient removal of N-linked carbohydrates from glycoproteins. Digestion speed and efficiency were compared to conventional in-solution based protocols. The use of PNGase F functionalized microcolumns resulted in efficient N-glycan removal in 10min from all major N-linked glycoprotein types of: (i) neutral (IgG), (ii) highly sialylated (fetuin), and (iii) high mannose (ribonuclease B) carbohydrate containing glycoprotein standards. The approach can be readily applied to automated sample preparation systems, such as liquid handling robots.
Implementation of an Automated High-Throughput Plasmid DNA Production Pipeline
Publication: Journal of Laboratory Automation, February 2016
Biologics sample management facilities are often responsible for a diversity of large-molecule reagent types, such as DNA, RNAi, and protein libraries. Historically, the management of large molecules was dispersed into multiple laboratories. As methodologies to support pathway discovery, antibody discovery, and protein production have become high throughput, the implementation of automation and centralized inventory management tools has become important. To this end, to improve sample tracking, throughput, and accuracy, we have implemented a module-based automation system integrated into inventory management software using multiple platforms (Hamilton, Hudson, Dynamic Devices, and Brooks). Here we describe the implementation of these systems with a focus on high-throughput plasmid DNA production management.
From haystack to needle: finding value with DNA encoded library technology at GSK
Publication: Med. Chem. Commun., Vol 11, 296-307, 2016
DNA encoded library technology (ELT) provides access to broad chemical diversity through affinity selection. As an early adopter, GlaxoSmithKline enabled the development of ELT from proof of concept to full fledged contributor to the small molecule therapeutic pipeline. Unique benefits of the GSK incubation include uptake of large numbers of building blocks and privileged scaffolds, guidance on library design, and opportunities to add value beyond lead discovery. Soluble epoxide hydrolase and RIP1 kinase represent two case studies of ELT hits that have advanced into clinical studies. ELT also shows promise as a strategy to forecast target tractability. As an emerging technology, it benefits from incubation at the interface of industry and academia.
Analysis of the Productivity of DNA Encoded Libraries
Publication: Med. Chem. Commun., 2016
DNA encoded library screens have gained recent interest as they allow for screening of millions of small molecules in a simple manner, with the goal of providing novel chemical starting points in target-based hit identification. Despite this interest, no publication describes the physical properties, novelty, or structural diversity of molecules derived from such screens, nor a comparison of productivity of different DNA encoded libraries. Here we address this gap by analysis of two DNA encoded library screens run against two protein targets employing mixtures of up to 16 different libraries. Fifty-seven exemplar small molecule compounds from 34 structurally distinct clusters were prioritized from the screening results, synthesized and tested for biochemical activity. Thirty-five of the 57 compounds possess significant biochemical activity (IC50 ≤ 10 μM). Seventeen of the 35 biochemically active compounds possess a molecular weight (MW) < 500 Dalton (Da) and clog P < 5, and 6 possess a MW < 400 Da and clog P < 4. None of the 57 DEL-derived compounds exist in the Roche corporate high throughput screening collection and public compound collections. Productivity per library was observed to be independent of library size. The most productive of the 16 investigated libraries was synthesized employing only simple chemistry. Physical properties of DEL-derived compounds correlate with average library properties when truncated sub-libraries are accounted for. Our analysis may help guide the design of future DNA encoded libraries.
Expression, purification, and characterization of recombinant human and murine milk fat globule-epidermal growth factor-factor 8
Publication: Protein Expression and Purification, Vol 124, 10-22, 2016
Milk fat globule-epidermal growth factor-factor 8 (MFG-E8), as its name suggests, is a major glycoprotein component of milk fat globules secreted by the mammary epithelium. Although its role in milk fat production is unclear, MFG-E8 has been shown to act as a bridge linking apoptotic cells to phagocytes for removal of these dying cells. MFG-E8 is capable of bridging these two very different cell types via interactions through both its epidermal growth factor (EGF)-like domain(s) and its lectin-type C domains. The EGF-like domain interacts with αVβ3 and αVβ5 integrins on the surface of phagocytes, whereas the C domains bind phosphatidylserine found on the surface of apoptotic cells. In an attempt to purify full-length, recombinant MFG-E8 expressed in either insect cells or CHO cells, we find that it is highly aggregated. Systematic truncation of the domain architecture of MFG-E8 indicates that the C domains are mainly responsible for the aggregation propensity. Addition of Triton X-100 to the conditioned cell culture media allowed partial recovery of non-aggregated, full-length MFG-E8. A more comprehensive detergent screen identified CHAPS as a stabilizer of MFG-E8 and allowed purification of a significant portion of non-aggregated, full-length protein. The CHAPS-stabilized recombinant MFG-E8 retained its natural ability to bind both αVβ3 and αVβ5 integrins and phosphatidylserine suggesting that it is properly folded and active. Herein we describe an efficient purification method for production of non-aggregated, full-length MFG-E8.
Automated screening for small organic ligands using DNA-encoded chemical libraries
Publication: Nature Protocols, Vol 11, No 4, 764-780, 2016
DNA-encoded chemical libraries (DECLs) are collections of organic compounds that are individually linked to different oligonucleotides, serving as amplifiable identification barcodes. As all compounds in the library can be identified by their DNA tags, they can be mixed and used in affinity-capture experiments on target proteins of interest. In this protocol, we describe the screening process that allows the identification of the few binding molecules within the multiplicity of library members. First, the automated affinity selection process physically isolates binding library members. Second, the DNA codes of the isolated binders are PCR-amplified and subjected to high-throughput DNA sequencing. Third, the obtained sequencing data are evaluated using a C++ program and the results are displayed using MATLAB software. The resulting selection fingerprints facilitate the discrimination of binding from nonbinding library members. The described procedures allow the identification of small organic ligands to biological targets from a DECL within 10 d.
The Type IV Pilus Assembly ATPase PilB of Myxococcus xanthus Interacts with the Inner Membrane Platform Protein PilC and the Nucleotide-binding Protein PilM
Publication: Journal of Biological Chemistry, No 6, 25, 2016
Type IV pili (T4P) are ubiquitous bacterial cell surface structures, involved in processes such as twitching motility, biofilm formation, bacteriophage infection, surface attachment, virulence, and natural transformation. T4P are assembled by machinery that can be divided into the outer membrane pore complex, the alignment complex that connects components in the inner and outer membrane, and the motor complex in the inner membrane and cytoplasm. Here, we characterize the inner membrane platform protein PilC, the cytosolic assembly ATPase PilB of the motor complex, and the cytosolic nucleotide-binding protein PilM of the alignment complex of the T4P machinery ofMyxococcus xanthus PilC was purified as a dimer and reconstituted into liposomes. PilB was isolated as a monomer and bound ATP in a non-cooperative manner, but PilB fused to Hcp1 ofPseudomonas aeruginosaformed a hexamer and bound ATP in a cooperative manner. Hexameric but not monomeric PilB bound to PilC reconstituted in liposomes, and this binding stimulated PilB ATPase activity. PilM could only be purified when it was stabilized by a fusion with a peptide corresponding to the first 16 amino acids of PilN, supporting an interaction between PilM and PilN(1-16). PilM-N(1-16) was isolated as a monomer that bound but did not hydrolyze ATP. PilM interacted directly with PilB, but only with PilC in the presence of PilB, suggesting an indirect interaction. We propose that PilB interacts with PilC and with PilM, thus establishing the connection between the alignment and the motor complex.
Evaluating high-throughput scale-down chromatography platforms for increased process understanding
Publication: Engineering in Life Sciences, 2016
In the biopharmaceutical industry, well-executed process development and characterization studies ensure robust manufacturing processes. In conventional chromatography, these studies are carried out in series with ≥10 mL bed volumes, thus requiring large quantities of feed material and operator oversight. For that reason, the screening of large process spaces becomes very expensive and has the potential to negatively impact other projects in a company’s portfolio competing for similar resources. In this study, we evaluated the ability of the three high-throughput process development formats 96-well filter plates, pipette tips, and mini columns to reduce resources in a late-phase process characterization Protein A capture step. The study used a Protein A capture step with a single experimental design, mAb feed material, and analytical package. The evaluation was based on how identical batch and dynamic process parameters impacted the quality and process performance attributes of monomer purity, host cell protein levels, and yield. All formats were able to provide similar models for product yield and monomer purity. Except for practical limitations of PreDictor plates, all formats could identify significant factors for host cell protein levels. RoboColumn units enabled dynamic factor evaluation and the results were the most comparable to conventional chromatography.
A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv)
Publication: Journal of Biological Chemistry, Vol 291, No 3, 1267-1276, 2016
Fully-human single-chain Fv (scFv) proteins are key potential building blocks of bispecific therapeutic antibodies, but they often suffer from manufacturability and clinical development limitations such as instability and aggregation. The causes of these scFv instability problems, in proteins that should be theoretically stable, remains poorly understood. To inform the future development of such molecules, we carried out a comprehensive structural analysis of the highly stabilized anti-CXCL13 scFv E10. E10 was derived from the parental 3B4 using complementarity-determining region (CDR)-restricted mutagenesis and tailored selection and screening strategies, and carries four mutations in VL-CDR3. High-resolution crystal structures of parental 3B4 and optimized E10 scFvs were solved in the presence and absence of human CXCL13. In parallel, a series of scFv mutants was generated to interrogate the individual contribution of each of the four mutations to stability and affinity improvements. In combination, these analyses demonstrated that the optimization of E10 was primarily mediated by removing clashes between both the VL and the VH, and between the VL and CXCL13. Importantly, a single, germline-encoded VL-CDR3 residue mediated the key difference between the stable and unstable forms of the scFv. This work demonstrates that, aside from being the critical mediators of specificity and affinity, CDRs may also be the primary drivers of biotherapeutic developability.
Lessons learned in building high throughput process development capabilities
Publication: Engineering in Life Sciences, 2016
Companies have adapted high-throughput techniques for process development to different levels. Some companies are beginning to incorporate high-throughput process development (HTPD) techniques at a level which is approachable for the beginner user (example: plate-based resin screening for purification) and others have sophisticated robotic platforms which are in routine use for new pipeline products for upstream, downstream, analytics, and just now starting in the protein formulation area. This mini review serves to summarize how companies have set up and are using HTPD capabilities, including the need for user-groups to improve the likelihood of success. The focus will be a practical summary of the utility and limitations of these approaches.
Current advances in the development of high-throughput purification strategies for the generation of therapeutic antibodies
Publication: Pharmaceutical Bioprocessing, Vol 3, No 6, 411-424, October 2015
The demand for therapeutic proteins, and particularly monoclonal antibodies, continues to grow within the biopharmaceutical industry. This is illustrated by the monoclonal antibody Humira® (adalimumab) having the highest sales of any drug in 2014. With the increasing numbers of biologics entering the market and the emergence of biosimilars and biobetters, costs need to be reduced in order for companies to remain competitive. High-throughput purification can provide the tools to meet the challenge of producing the thousands of potential drug candidates generated in drug discovery while simultaneously providing robust and cost-effective purification strategies in both research and production. In this review, we discuss the many different methodologies and automation technologies that can be employed in high-throughput purification.
DNA Encoded Library Selections and Insights Provided by Computational Simulations
Publication: ACS Chemical Biology, July 2015
DNA encoded library (DEL) technology allows for rapid generation of extremely large numbers of small molecules and is often used to find novel chemical starting points for pharmaceutically relevant proteins. DEL selection output consists of a list of small-molecule structures and enrichment levels. It is widely presumed that molecules with greater enrichment will have larger equilibrium association constants, and follow-up efforts are triaged accordingly. Herein we describe a simple mathematical model used to simulate DEL selections. Simulations predict that enrichment levels will correlate poorly with equilibrium association constants when selections use high concentrations of protein or lower quality DELs (high variance in final product synthetic yields). A potentially superior technique is demonstrated to qualitatively assess equilibrium association constants directly from sequencing data. This technique requires conducting selections over a range of protein concentrations, so that the influence of synthetic yield can be accounted for.
High-throughput purification tools for rapid upstream process development are interchangeable for biologics
Publication: Engineering in Life Sciences, July 2015
Upstream process development of biologics is not only productivity-driven but also quality-driven. Typically, most quality attributes are not directly measurable in cell culture samples due to low product concentration and purity, thus requiring some level of sample purification. As higher throughput upstream technologies become available, sample purification is becoming a bottleneck in limiting the number and types of cell culture samples that can be analyzed. The application of high-throughput, microscale protein purification techniques has the potential to address and expand this capability. In this work, the affinity capture step of an IgG1 mAb was adapted to fit resin-plate, resin-tip, and mini-column formats in an attempt to approximate the packed-column performance by optimizing parameters such as contact time, liquid/resin ratio, and loading to produce a yield of ≥70% yield. A representative cell culture supernatant was purified using both the optimized microscale and conventional techniques, and analyzed using a comprehensive panel of product quality assays. This direct comparison demonstrated that each technique generates product of equivalent purity across a wide range of feed conditions. The analytical comparability suggests that any of the conventional and high-throughput methods are interchangeable for biologics, allowing flexible development of an end-to-end integrated high-throughput strategy.
Improved assembly of bispecific antibodies from knob and hole half-antibodies
Publication: Biotechnology Progress, July 2015
A process was developed for large-scale assembly of IgG1 and IgG4 bispecific antibodies from knob and hole half-antibodies. We optimized assembly conditions such as pH, temperature, stabilizers, and reducing agent. We also identified and exploited structural changes unique to knob and hole half-antibodies with the result of improving assembly outcome, specifically storing half-antibodies at higher pH will condition them to assemble more rapidly and produce fewer high molecular-weight species (HMWS). Application of heat to the assemblies resulted in an acceleration of assembly rate, with optimal formation of bispecific achieved at 37°C. IgG4 half-antibodies were unusually sensitive to temperature-dependent formation of HMWS in pre-assembly conditioning as well as during assembly. We selected l-histidine and Polyvinylpyrrolidone (PVP) as stabilizers to prevent HMWS formation in IgG4 , and achieved rapid and high-efficiency assemblies. Using optimized assembly conditions, we developed and scaled up a method for assembling bispecific antibody with 90% assembly efficiency over 6 h with minimal impact to product quality, generating a pool with bispecific antibody for downstream processing.
Comparative glycoprofiling of HIV gp120 immunogens by capillary electrophoresis and MALDI mass spectrometry
Publication: Electrophoresis, Vol 36, No 11-12, 1305-1313, June 2015
The human immunodeficiency virus (HIV) envelope glycoprotein (Env) is the primary antigenic feature on the surface of the virus and is of key importance in HIV vaccinology. Vaccine trials with the gp120 subunit of Env are ongoing, with the recent RV144 trial showing moderate efficacy. gp120 is densely covered with N-linked glycans that are thought to help evade the host’s humoral immune response. To assess how the global glycosylation patterns vary between gp120 constructs, the glycan profiles of several gp120s were examined by CE with LIF detection and MALDI-MS. The glycosylation profiles were found to be similar for chronic versus transmitter/founder isolates and only varied moderately between gp120s from different clades. This study revealed that the addition of specific tags, such as the herpes simplex virus glycoprotein D tag used in the RV144 trial, had significant effects on the overall glycosylation patterns. Such effects are likely to influence the immunogenicity of various Env immunogens and should be considered for future vaccine strategies, emphasizing the importance of the glycosylation analysis approach described in this paper.
Encoded library technology screening of hepatitis C virus NS4B yields a small-molecule compound series with in vitro replicon activity
Publication: Antimicrobial agents and chemotherapy, Vol 59, No 6, 3450-3459, June 2015
To identify novel antivirals to the hepatitis C virus (HCV) NS4B protein, we utilized encoded library technology (ELT), which enables purified proteins not amenable to standard biochemical screening methods to be tested against large combinatorial libraries in a short period of time. We tested NS4B against several DNA-encoded combinatorial libraries (DEL) and identified a single DEL feature that was subsequently progressed to off-DNA synthesis. The most active of the initial synthesized compounds had 50% inhibitory concentrations (IC50s) of 50 to 130 nM in a NS4B radioligand binding assay and 300 to 500 nM in an HCV replicon assay. Chemical optimization yielded compounds with potencies as low as 20 nM in an HCV genotype 1b replicon assay, 500 nM against genotype 1a, and 5 μM against genotype 2a. Through testing against other genotypes and genotype 2a-1b chimeric replicons and from resistance passage using the genotype 1b replicon, we confirmed that these compounds were acting on the proposed first transmembrane region of NS4B. A single sequence change (F98L) was identified as responsible for resistance, and it was thought to largely explain the relative lack of potency of this series against genotype 2a. Unlike other published series that appear to interact with this region, we did not observe sensitivity to amino acid substitutions at positions 94 and 105. The discovery of this novel compound series highlights ELT as a valuable approach for identifying direct-acting antivirals to nonenzymatic targets.
Poly(N-vinylimidazole/ethylene glycol dimethacrylate) for the purification and isolation of phenolic acids
Publication: Analytica Chimica Acta, Vol 885, 199-206, June 2015
In this study we report the novel polymeric resin poly(N-vinyl imidazole/ethylene glycol dimethacrylate) for the purification and isolation of phenolic acids. The monomer to crosslinker ratio and the porogen composition were optimized for isolating phenolic acids diluted in acetonitrile at normal phase chromatography conditions, first. Acetonitrile serves as polar, aprotic solvent, dissolving phenolic acids but not interrupting interactions with the stationary phase due to the approved Hansen solubility parameters. The optimized resin demonstrated high loading capacities and adsorption abilities particularly for phenolic acids in both, acetonitrile and aqueous solutions. The adsorption behavior of aqueous standards can be attributed to ion exchange effects due to electrostatic interactions between protonated imidazole residues and deprotonated phenolic acids. Furthermore, adsorption experiments and subsequent curve fittings provide information of maximum loading capacities of single standards according to the Langmuir adsorption model. Recovery studies of the optimized polymer in the normal-phase and ion-exchange mode illustrate the powerful isolation properties for phenolic acids and are comparable or even better than typical, commercially available solid phase extraction materials. In order to prove the applicability, a highly complex extract of rosemary leaves was purified by poly(N-vinyl imidazole/ethylene glycol dimethacrylate) and the isolated compounds were identified using UHPLC-qTOF-MS.
Use of the site-specific retargeting jump-in platform cell line to support biologic drug discovery
Publication: Journal of biomolecular screening, Vol 20, No 4, 428-535, April 2015
Biologics represent a fast-growing class of therapeutics in the pharmaceutical sector. Discovery of therapeutic antibodies and characterization of peptides can necessitate high expression of the target gene requiring the generation of clonal stably transfected cell lines. Traditional challenges of stable cell line transfection include gene silencing and cell-to-cell variability. Our inability to control these can present challenges in lead isolation. Recent progress in site-specific targeting of transgene to specific genomic loci has transformed the ability to generate stably transfected mammalian cell lines. In this article, we describe how the use of the Jump-In platform (Life Technologies, Carlsbad, CA) has been applied to drug discovery projects. It can easily and rapidly generate homogeneous high-expressing cell pools with a high degree of reproducibility. Their use in cell-based screening to identify specific binders, identify binding to relevant species variants, or detect functionally relevant therapeutic antibodies is central in driving drug discovery.
A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell-free protein synthesis systems
Publication: Biotechnology Progress, Vol 31, No 3, 823-831, 2015
Cell-free protein synthesis (CFPS) systems allow for robust protein expression with easy manipulation of conditions to improve protein yield and folding. Recent technological developments have significantly increased the productivity and reduced the operating costs of CFPS systems, such that they can compete with conventional in vivo protein production platforms, while also offering new routes for the discovery and production of biotherapeutics. As cell-free systems have evolved, productivity increases have commonly been obtained by addition of components to previously designed reaction mixtures without careful re-examination of the essentiality of reagents from previous generations. Here we present a systematic sensitivity analysis of the components in a conventional Escherichia coli CFPS reaction mixture to evaluate their optimal concentrations for production of the immunoglobulin G trastuzumab. We identify eight changes to the system, which result in optimal expression of trastuzumab. We find that doubling the potassium glutamate concentration, while entirely eliminating pyruvate, coenzyme A, NAD, total tRNA, folinic acid, putrescine and ammonium glutamate, results in a highly productive cell-free system with a 95% reduction in reagent costs (excluding cell-extract, plasmid, and T7 RNA polymerase made in-house). A larger panel of other proteins was also tested and all show equivalent or improved yields with our simplified system. Furthermore, we demonstrate that all of the reagents for CFPS can be combined in a single freeze-thaw stable master mix to improve reliability and ease of use. These improvements are important for the application of the CFPS system in fields such as protein engineering, high-throughput screening, and biotherapeutics.
Sample Preparation for N-Glycosylation Analysis of Therapeutic Monoclonal Antibodies by Electrophoresis
Publication: Methods in Molecular Biology, Vol 1274, 183-195, 2015
There are a considerable number of biopharmaceuticals that have been approved for clinical use in the past decade. Over half of these new generation drugs are glycoproteins, such as monoclonal antibodies or other recombinant glycoproteins, which are mostly produced in mammalian cell lines. The linked carbohydrate moieties affect not only their physicochemical properties and thermal stability but also crucial features like receptor-binding activity, circulating half-life, as well as immunogenicity. The structural diversity of these attached glycans can be manifested in altered monosaccharide composition and linkages/positions among the monosaccharide building blocks. In addition, as more and more biosimilar products hit the market, understanding the effects of their glycosylation modification has become a recent target in efficacy and safety issues. To ensure consistent quality of these products, glycosylation profiles have to be monitored and controlled in all steps of the manufacturing process, i.e., from clone selection to lot release. In this paper, we describe some of the recently introduced and commonly used sample preparation techniques for capillary electrophoresis (CE)-based profiling and structural elucidation of N-glycans. The presented protocols include protein A affinity partitioning of monoclonal antibodies (mAbs), enzymatic release of the N-linked glycans, labeling of the liberated carbohydrates, reaction mixture purification techniques to remove the excess labeling reagent, and high-resolution and rapid capillary electrophoresis-laser-induced fluorescence (CE-LIF)-based profiling of the labeled and purified N-glycans.
Generation of potent mouse monoclonal antibodies to self-proteins using T-cell epitope “tags.”
Augmented Binary Substitution: Single-pass CDR germ-lining and stabilization of therapeutic antibodies
Publication: Proceedings of the National Academy of Sciences of the United States of America, Vol 112, No 50, 15354-15359, 2015
Although humanized antibodies have been highly successful in the clinic, all current humanization techniques have potential limitations, such as: reliance on rodent hosts, immunogenicity due to high non-germ-line amino acid content, v-domain destabilization, expression and formulation issues. This study presents a technology that generates stable, soluble, ultrahumanized antibodies via single-step complementarity-determining region (CDR) germ-lining. For three antibodies from three separate key immune host species, binary substitution CDR cassettes were inserted into preferred human frameworks to form libraries in which only the parental or human germ-line destination residue was encoded at each position. The CDR-H3 in each case was also augmented with 1 ± 1 random substitution per clone. Each library was then screened for clones with restored antigen binding capacity. Lead ultrahumanized clones demonstrated high stability, with affinity and specificity equivalent to, or better than, the parental IgG. Critically, this was mainly achieved on germ-line frameworks by simultaneously subtracting up to 19 redundant non-germ-line residues in the CDRs. This process significantly lowered non-germ-line sequence content, minimized immunogenicity risk in the final molecules and provided a heat map for the essential non-germ-line CDR residue content of each antibody. The ABS technology therefore fully optimizes the clinical potential of antibodies from rodents and alternative immune hosts, rendering them indistinguishable from fully human in a simple, single-pass process.
Optimisation of a simple method to transiently transfect a CHO cell line in high-throughput and at large scale
Publication: Protein Expression and Purification, 2015
Transient expression of heterologous proteins in mammalian systems is a powerful way to generate protein reagents quickly. However, it has historically suffered from poor yields in comparison to methods where the recombinant gene is stably integrated into the genome and high expressing clones isolated. Transient methods have been well described for HEK-based systems. In this paper we show the use of a design of experiments (DoE) approach to quickly analyse the effect of a range of different parameters on protein expression from a CHO-based transient system. We show that this system is amenable to a very simple transfection procedure by independent direct addition of DNA and transfection reagent to the culture vessel. In addition we show that expression can be improved by reducing the temperature of the culture conditions post-transfection. The process is demonstrated to be transferrable from 3 ml cultures in deep 24-well plates through cultures in CultiFlask Bioreactors, shake flasks and up to 25 L culture in Wave Bioreactors. Data are shown to illustrate the utility of the system with a number of different classes of protein.
Structure of a highly acidic β-lactamase from the moderate halophile Chromohalobacter sp. 560 and the discovery of a Cs(+)-selective binding site
Publication: Acta Crystallographica Section D: Biological Crystallography, Vol 71, No 3, 541-554, 2015
Environmentally friendly absorbents are needed for Sr(2+) and Cs(+), as the removal of the radioactive Sr(2+) and Cs(+) that has leaked from the Fukushima Nuclear Power Plant is one of the most important problems in Japan. Halophilic proteins are known to have many acidicresidues on their surface that can provide specific binding sites for metal ions such as Cs(+) or Sr(2+). The crystal structure of a halophilic β-lactamase from Chromohalobacter sp. 560(HaBLA) was determined to resolutions of between 1.8 and 2.9 Å in space group P31 using X-ray crystallography. Moreover, the locations of bound Sr(2+) and Cs(+) ions were identified by anomalous X-ray diffraction. The location of one Cs(+)-specific binding site was identified in HaBLA even in the presence of a ninefold molar excess of Na(+) (90 mM Na(+)/10 mM Cs(+)). From an activity assay using isothermal titration calorimetry, the bound Sr(2+) and Cs(+) ions do not significantly affect the enzymatic function of HaBLA. The observation of a selective and high-affinity Cs(+)-binding site provides important information that is useful for the design of artificial Cs(+)-binding sites that may be useful in the bioremediation of radioactive isotopes
Automated pipeline for rapid production and screening of HIV-specific monoclonal antibodies using pichia pastoris
Publication: Biotechnology and Bioengineering, 2015
Monoclonal antibodies (mAbs) that bind and neutralize human pathogens have great therapeutic potential. Advances in automated screening and liquid handling have resulted in the ability to discover antigen-specific antibodies either directly from human blood or from various combinatorial libraries (phage, bacteria, or yeast). There remain, however, bottlenecks in the cloning, expression and evaluation of such lead antibodies identified in primary screens that hinder high-throughput screening. As such, “hit-to-lead identification” remains both expensive and time-consuming. By combining the advantages of overlap extension PCR (OE-PCR) and a genetically stable yet easily manipulatable microbial expression host Pichia pastoris, we have developed an automated pipeline for the rapid production and screening of full-length antigen-specific mAbs. Here, we demonstrate the speed, feasibility and cost-effectiveness of our approach by generating several broadly neutralizing antibodies against human immunodeficiency virus (HIV).
Identification of anti-tumour biologics using primary tumour models, 3-D phenotypic screening and image-based multi-parametric profiling
Publication: Molecular Cancer, Vol 14, No 1, 147, 2015
BACKGROUND: Monolayer cultures of immortalised cell lines are a popular screening tool for novel anti-cancer therapeutics, but these methods can be a poor surrogate for disease states, and there is a need for drug screening platforms which are more predictive of clinical outcome. In this study, we describe a phenotypic antibody screen using three-dimensional cultures of primary cells, and image-based multi-parametric profiling in PC-3 cells, to identify anti-cancer biologics against new therapeutic targets.
METHODS: ScFv Antibodies and designed ankyrin repeat proteins (DARPins) were isolated using phage display selections against primary non-small cell lung carcinoma cells. The selected molecules were screened for anti-proliferative and pro-apoptotic activity against primary cells grown in three-dimensional culture, and in an ultra-high content screen on a 3-D cultured cell line using multi-parametric profiling to detect treatment-induced phenotypic changes. The targets of molecules of interest were identified using a cell-surface membrane protein array. An anti-CUB domain containing protein 1 (CDCP1) antibody was tested for tumour growth inhibition in a patient-derived xenograft model, generated from a stage-IV non-small cell lung carcinoma, with and without cisplatin.
RESULTS: Two primary non-small cell lung carcinoma cell models were established for antibody isolation and primary screening in anti-proliferative and apoptosis assays. These assays identified multiple antibodies demonstrating activity in specific culture formats. A subset of the DARPins was profiled in an ultra-high content multi-parametric screen, where 300 morphological features were measured per sample. Machine learning was used to select features to classify treatment responses, then antibodies were characterised based on the phenotypes that they induced. This method co-classified several DARPins that targeted CDCP1 into two sets with different phenotypes. Finally, an anti-CDCP1 antibody significantly enhanced the efficacy of cisplatin in a patient-derived NSCLC xenograft model.
CONCLUSIONS: Phenotypic profiling using complex 3-D cell cultures steers hit selection towards more relevant in vivo phenotypes, and may shed light on subtle mechanistic variations in drug candidates, enabling data-driven decisions for oncology target validation. CDCP1 was identified as a potential target for cisplatin combination therapy.
Arginine methylation and citrullination of splicing factor proline- and glutamine-rich (SFPQ/PSF) regulates its association with mRNA
Publication: RNA, Vol 21, No 3, 1-13, 2015
Splicing factor proline- and glutamine-rich (SFPQ) also commonly known as polypyrimidine tract-binding protein-associated-splicing factor (PSF) and its binding partner non-POU domain-containing octamer-binding protein (NONO/p54nrb), are highly abundant, multifunctional nuclear proteins. However, the exact role of this complex is yet to be determined. Following purification of the endogeneous SFPQ/NONO complex, mass spectrometry analysis identified a wide range of interacting proteins, including those involved in RNA processing, RNA splicing, and transcriptional regulation, consistent with a multifunctional role for SFPQ/NONO. In addition, we have identified several sites of arginine methylation in SFPQ/PSF using mass spectrometry and found that several arginines in the N-terminal domain of SFPQ/PSF are asymmetrically dimethylated. Furthermore, we find that the protein arginine N-methyltransferase, PRMT1, catalyzes this methylation in vitro and that this is antagonized by citrullination of SFPQ. Arginine methylation and citrullination of SFPQ/PSF does not affect complex formation with NONO. However, arginine methylation was shown to increase the association with mRNA in mRNP complexes in mammalian cells. Finally we show that the biochemical properties of the endogenous complex from cell lysates are significantly influenced by the ionic strength during purification. At low ionic strength, the SFPQ/NONO complex forms large heterogeneous protein assemblies or aggregates, preventing the purification of the SFPQ/NONO complex. The ability of the SFPQ/NONO complex to form varying protein assemblies, in conjunction with the effect of post-translational modifications of SFPQ modulating mRNA binding, suggests key roles affecting mRNP dynamics within the cell.
Generation of novel monoclonal antibodies for the enrichment and characterization of human corneal endothelial cells (hCENC) necessary for the treatment of corneal endothelial blindness
Publication: mAbs, Vol 6, 1439-1452, November 2014
Corneal transplantation is the primary treatment option to restore vision for patients with corneal endothelial blindness. Although the success rate of treatment is high, limited availability of transplant grade corneas is a major obstacle. Tissue-engineered corneal endothelial grafts constructed using cultivated human corneal endothelial cells (hCENC) isolated from cadaveric corneas may serve as a potential graft source. Currently, tools for the characterization of cultured hCENC and enrichment of hCENC from potential contaminating cells such as stromal fibroblasts are lacking. In this study, we describe the generation and characterization of novel cell surface monoclonal antibodies (mAbs) specific for hCENC. These mAbs could be used for enrichment and characterization of hCENC. Out of a total of 389 hybridomas, TAG-1A3 and TAG-2A12 were found to be specific to the corneal endothelial monolayer by immunostaining of frozen tissue sections. Both mAbs were able to clearly identify hCENC with good ‘cobblestone-like’ morphology from multiple donors. The antigen targets for TAG-1A3 and TAG-2A12 were found to be CD166/ALCAM and Peroxiredoxin-6 (Prdx-6), respectively, both of which have not been previously described as markers of hCENC. Additionally, unlike other Prdx-6 mAbs, TAG-2A12 was found to specifically bind cell surface Prdx-6, which was only expressed on hCENC and not on other cell types screened such as human corneal stromal fibroblasts (hCSF) and human pluripotent stem cells (hPSC). From our studies, we conclude that TAG-1A3 and TAG-2A12 are promising tools to quantitatively assess hCENC quality. It is also noteworthy that the binding specificity of TAG-2A12 could be used for the enrichment of hCENC from cell mixtures of hCSF and hPSC.
Production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system
Publication: Bioconjugate Chemistry, Vol 25, No 2, 351-361, February 2014
Antibody-drug conjugates (ADCs) are a targeted chemotherapeutic currently at the cutting edge of oncology medicine. These hybrid molecules consist of a tumor antigen-specific antibody coupled to a chemotherapeutic small molecule. Through targeted delivery of potent cytotoxins, ADCs exhibit improved therapeutic index and enhanced efficacy relative to traditional chemotherapies and monoclonal antibody therapies. The currently FDA-approved ADCs, Kadcyla (Immunogen/Roche) and Adcetris (Seattle Genetics), are produced by conjugation to surface-exposed lysines, or partial disulfide reduction and conjugation to free cysteines, respectively. These stochastic modes of conjugation lead to heterogeneous drug products with varied numbers of drugs conjugated across several possible sites. As a consequence, the field has limited understanding of the relationships between the site and extent of drug loading and ADC attributes such as efficacy, safety, pharmacokinetics, and immunogenicity. A robust platform for rapid production of ADCs with defined and uniform sites of drug conjugation would enable such studies. We have established a cell-free protein expression system for production of antibody drug conjugates through site-specific incorporation of the optimized non-natural amino acid, para-azidomethyl-l-phenylalanine (pAMF). By using our cell-free protein synthesis platform to directly screen a library of aaRS variants, we have discovered a novel variant of the Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS), with a high activity and specificity toward pAMF. We demonstrate that site-specific incorporation of pAMF facilitates near complete conjugation of a DBCO-PEG-monomethyl auristatin (DBCO-PEG-MMAF) drug to the tumor-specific, Her2-binding IgG Trastuzumab using strain-promoted azide-alkyne cycloaddition (SPAAC) copper-free click chemistry. The resultant ADCs proved highly potent in in vitro cell cytotoxicity assays.
Recent advances in the analysis of therapeutic proteins by capillary and microchip electrophoresis
Publication: Anal Methods., Vol 6, No 15, 5427-49, 2014
The development of therapeutic proteins and peptides is an expensive and time-intensive process. Biologics, which have become a multi-billion dollar industry, are chemically complex products that require constant observation during each stage of development and production. Post-translational modifications along with chemical and physical degradation from oxidation, deamidation, and aggregation, lead to high levels of heterogeneity that affect drug quality and efficacy. The various separation modes of capillary electrophoresis (CE) are commonly utilized to perform quality control and assess protein heterogeneity. This review attempts to highlight the most recent developments and applications of CE separation techniques for the characterization of protein and peptide therapeutics by focusing on papers accepted for publication in the in the two-year period between January 2012 and December 2013. The separation principles and technological advances of CE, capillary gel electrophoresis, capillary isoelectric focusing, capillary electrochromatography and CE-mass spectrometry are discussed, along with exciting new applications of these techniques to relevant pharmaceutical issues. Also included is a small selection of papers on microchip electrophoresis to show the direction this field is moving with regards to the development of inexpensive and portable analysis systems for on-site, high-throughput analysis.
Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes
Publication: mAbs, Vol 6, No 6, 1425-1438, 2014
Generation of functional antibodies against integral membrane proteins such as the G-protein coupled receptor CXCR2 is technically challenging for several reasons, including limited epitope accessibility, the requirement for a lipid environment to maintain structure and their existence in dynamic conformational states. Antibodies to human CXCR2 were generated by immunization in vivo and by in vitro selection methods. Whole cell immunization of transgenic mice and screening of phage display libraries using CXCR2 magnetic proteoliposomes resulted in the isolation of antibodies with distinct modes of action. The hybridoma-derived antibody fully inhibited IL-8 and Gro-α responses in calcium flux and β-arrestin recruitment assays. The phage-display derived antibodies were allosteric antagonists that showed ligand dependent differences in functional assays. The hybridoma and phage display antibodies did not cross-compete in epitope competition assays and mapping using linear and CLIPS peptides confirmed that they recognized distinct epitopes of human CXCR2. This illustrates the benefits of using parallel antibody isolation approaches with different antigen presentation methods to successfully generate functionally and mechanistically diverse antagonistic antibodies to human CXCR2. The method is likely to be broadly applicable to other complex membrane proteins.
Encoded library technology as a source of hits for the discovery and lead optimization of a potent and selective class of bactericidal direct inhibitors of Mycobacterium tuberculosis InhA
Publication: Journal of Medicinal Chemistry, Vol 57, No 4, 1276-1288, 2014
Tuberculosis (TB) is one of the world’s oldest and deadliest diseases, killing a person every 20 s. InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis, is the target of the frontline antitubercular drug isoniazid (INH). Compounds that directly target InhA and do not require activation by mycobacterial catalase peroxidase KatG are promising candidates for treating infections caused by INH resistant strains. The application of the encoded library technology (ELT) to the discovery of direct InhA inhibitors yielded compound 7 endowed with good enzymatic potency but with low antitubercular potency. This work reports the hit identification, the selected strategy for potency optimization, the structure-activity relationships of a hundred analogues synthesized, and the results of the in vivo efficacy studies performed with the lead compound 65.
Remediating agitation-induced antibody aggregation by eradicating exposed hydrophobic motifs
Publication: mAbs, Vol 6, No 6, 1540-1550, 2014
Therapeutic antibodies must encompass drug product suitable attributes to be commercially marketed. An undesirable antibody characteristic is the propensity to aggregate. Although there are computational algorithms that predict the propensity of a protein to aggregate from sequence information alone, few consider the relevance of the native structure. The Spatial Aggregation Propensity (SAP) algorithm developed by Chennamsetty et. al. incorporates structural and sequence information to identify motifs that contribute to protein aggregation. We have utilized the algorithm to design variants of a highly aggregation prone IgG2. All variants were tested in a variety of high-throughput, small-scale assays to assess the utility of the method described herein. Many variants exhibited improved aggregation stability whether induced by agitation or thermal stress while still retaining bioactivity.
Galactosyltransferase 4 is a major control point for glycan branching in N-linked glycosylation
Strategy for selecting disposable bags for cell culture media applications based on a root-cause investigation
Publication: Biotechnology Progress, Vol 29, No 6, 1535-1549, November 2013
The use of disposable bags for cell culture media storage has grown significantly in the past decade. Some of the key advantages of using disposable bags relative to non-disposable containers include increased product throughput, decreased cleaning validation costs, reduced risk of cross contamination and lower facility costs. As the scope of use of disposable bags for cell culture applications increases, problematic bags and scenarios should be identified and addressed to continue improving disposables technologies and meet the biotech industry’s needs. In this article, we examine a cell culture application wherein media stored in disposable bags is warmed at 37°C before use for cell culture operations. A problematic bag film was identified through a prospective and retrospective cell culture investigation. The investigation provided information on the scope and variation of the issue with respect to different Chinese hamster ovary (CHO) cell lines, cell culture media, and application-specific parameters. It also led to the development of application-specific test methods and enabled a strategy for disposable bag film testing. The strategy was implemented for qualifying an alternative bag film for use in our processes. In this test strategy, multiple lots of 13 bag film types, encompassing eight vendors were evaluated using a three round, cell culture-based test strategy. The test strategy resulted in the determination of four viable bag film options based on the technical data. The results of this evaluation were used to conclude that a volatile or air-quenched compound, likely generated by gamma irradiation of the problematic bag film, negatively impacted cell culture performance.
The impact of microcarrier culture optimization on the glycosylation profile of a monoclonal antibody
Publication: SpringerPlus, Vol 2, No 1, 25, 2013
Microcarriers are widely used for the large-scale culture of attachment-dependent cells with increased cell densities and, ultimately, higher product yield. In these processes, the specific culture conditions can affect the quality of the product, which is closely related to its glycosylation pattern. Furthermore, the lack of studies in the area reinforces the need to better understand the effects of microcarrier culture in product glycosylation. Consequently, in this work, the glycosylation profile of a monoclonal antibody (mAb) produced by adherent CHO-K1 cells grown in Cytodex 3 was evaluated under different conditions, and compared to that obtained of typical adherent cultures. It was found that microcarrier cultures result in a glycosylation profile with different characteristics from T-flask cultures, with a general increase in galactosylation and decrease in fucosylation levels, both with a potentially positive impact on mAb activity. Sialylation also varied but without a general tendency. This study then showed that the specific culture conditions used in microcarrier culture influence the mAb glycan profile, and each functional element (galactose, core fucose, sialic acid) is independently affected by these conditions. In particular, great reductions of fucosylation (from 79 to 55%) were obtained when using half volume at inoculation, and notable decreases in sialylation (from 23 to 2%) and glycoform heterogeneity (from 20 to 11 glycoforms) were observed for shake flask culture, potentially associated with the improved cell densities achieved in these culture vessels.
Combining phenotypic and proteomic approaches to identify membrane targets in a ‘triple negative’ breast cancer cell type
Publication: Molecular cancer, Vol 12, No 1, 11, 2013
BACKGROUND: The continued discovery of therapeutic antibodies, which address unmet medical needs, requires the continued discovery of tractable antibody targets. Multiple protein-level target discovery approaches are available and these can be used in combination to extensively survey relevant cell membranomes. In this study, the MDA-MB-231 cell line was selected for membranome survey as it is a ‘triple negative’ breast cancer cell line, which represents a cancer subtype that is aggressive and has few treatment options.
METHODS: The MDA-MB-231 breast carcinoma cell line was used to explore three membranome target discovery approaches, which were used in parallel to cross-validate the significance of identified antigens. A proteomic approach, which used membrane protein enrichment followed by protein identification by mass spectrometry, was used alongside two phenotypic antibody screening approaches. The first phenotypic screening approach was based on hybridoma technology and the second was based on phage display technology. Antibodies isolated by the phenotypic approaches were tested for cell specificity as well as internalisation and the targets identified were compared to each other as well as those identified by the proteomic approach. An anti-CD73 antibody derived from the phage display-based phenotypic approach was tested for binding to other ‘triple negative’ breast cancer cell lines and tested for tumour growth inhibitory activity in a MDA-MB-231 xenograft model.
RESULTS: All of the approaches identified multiple cell surface markers, including integrins, CD44, EGFR, CD71, galectin-3, CD73 and BCAM, some of which had been previously confirmed as being tractable to antibody therapy. In total, 40 cell surface markers were identified for further study. In addition to cell surface marker identification, the phenotypic antibody screening approaches provided reagent antibodies for target validation studies. This is illustrated using the anti-CD73 antibody, which bound other ‘triple negative’ breast cancer cell lines and produced significant tumour growth inhibitory activity in a MDA-MB-231 xenograft model.
CONCLUSIONS: This study has demonstrated that multiple methods are required to successfully analyse the membranome of a desired cell type. It has also successfully demonstrated that phenotypic antibody screening provides a mechanism for rapidly discovering and evaluating antibody tractable targets, which can significantly accelerate the therapeutic discovery process.
CDR-restricted engineering of native human scFvs creates highly stable and soluble bifunctional antibodies for subcutaneous delivery
Publication: mAbs, Vol 5, No 6, 882-895, 2013
While myriad molecular formats for bispecific antibodies have been examined to date, the simplest structures are often based on the scFv. Issues with stability and manufacturability in scFv-based bispecific molecules, however, have been a significant hindrance to their development, particularly for high-concentration, stable formulations that allow subcutaneous delivery. Our aim was to generate a tetravalent bispecific molecule targeting two inflammatory mediators for synergistic immune modulation. We focused on an scFv-Fc-scFv format, with a flexible (A4T)3 linker coupling an additional scFv to the C-terminus of an scFv-Fc. While one of the lead scFvs isolated directly from a naïve library was well-behaved and sufficiently potent, the parental anti-CXCL13 scFv 3B4 required optimization for affinity, stability, and cynomolgus ortholog cross-reactivity. To achieve this, we eschewed framework-based stabilizing mutations in favor of complementarity-determining region (CDR) mutagenesis and re-selection for simultaneous improvements in both affinity and thermal stability. Phage-displayed 3B4 CDR-mutant libraries were used in an aggressive “hammer-hug” selection strategy that incorporated thermal challenge, functional, and biophysical screening. This approach identified leads with improved stability and>18-fold, and 4,100-fold higher affinity for both human and cynomolgus CXCL13, respectively. Improvements were exclusively mediated through only 4 mutations in VL-CDR3. Lead scFvs were reformatted into scFv-Fc-scFvs and their biophysical properties ranked. Our final candidate could be formulated in a standard biopharmaceutical platform buffer at 100 mg/ml with<2% high molecular weight species present after 7 weeks at 4 °C and viscosity<15 cP. This workflow has facilitated the identification of a truly manufacturable scFv-based bispecific therapeutic suitable for subcutaneous administration.
Automated sample preparation for CE-SDS
Publication: Electrophoresis, Vol 34, No 9-10, 1369-1374, 2013
Traditionally, CE with SDS (CE-SDS) places many restrictions on sample composition. Requirements include low salt content, known initial sample concentration, and a narrow window of final sample concentration. As these restrictions require buffer exchange for many sample types, sample preparation is often tedious and yields poor sample recoveries. To improve capacity and streamline sample preparation, an automated robotic platform was developed using the PhyNexus Micro-Extractor Automated Instrument (MEA) for both the reduced and nonreduced CE-SDS assays. This automated sample preparation normalizes sample concentration, removes salts and other contaminants, and adds the required CE-SDS reagents, essentially eliminating manual steps during sample preparation. Fc-fusion proteins and monoclonal antibodies were used in this work to demonstrate benefits of this approach when compared to the manual method. With optimized conditions, this application has demonstrated decreased analyst “hands on” time and reduced total assay time. Sample recovery greater than 90% can be achieved, regardless of initial composition and concentration of analyte.
Facile fabrication and instant application of miniaturized antibody-decorated affinity columns for higher-order structure and functional characterization of TRIM21 epitope peptides
Publication: Analytical Chemistry, Vol 85, No 21, 10479-10487, 2013
Both epitope excision and epitope extraction methods, combined with mass spectrometry, generate precise informations on binding surfaces of full-length proteins, identifying sequential (linear) or assembled (conformational) epitopes, respectively. Here, we describe the one-step fabrication and application of affinity columns using reversibly immobilized antibodies with highest flexibility with respect to antibody sources and lowest sample amount requirements (fmol range). Depending on the antibody source, we made use of protein G- or protein A-coated resins as support materials. These materials are packed in pipet tips and in combination with a programmable multichannel pipet form a highly efficient epitope mapping system. In addition to epitope identification, the influence of epitope structure modifications on antibody binding specificities could be studied in detail with synthetic peptides. Elution of epitope peptides was optimized such that mass spectrometric analysis was feasible after a single desalting step. Epitope peptides were identified by accurate molecular mass determinations or by partial amino acid sequence analysis. In addition, charge state comparison or ion mobility analysis of eluted epitope peptides enabled investigation of higher-order structures. The epitope peptide of the TRIM21 (TRIM: tripartite motif) autoantigen that is recognized by a polyclonal antibody was determined as assembling an “L-E-Q-L” motif on an α-helix. Secondary structure determination by circular dichroism spectroscopy and structure modeling are in accordance with the mass spectrometric results and the antigenic behavior of the 17-mer epitope peptide variants from the full-length autoantigen.
High-throughput process development of chromatography steps: advantages and limitations of different formats used
Publication: Biotechnology Journal, Vol 7, No 10, 1192-1202, October 2012
In the past, development of a chromatographic separation method has been accomplished by performing a series of experiments using either manual or automated chromatography systems. The screening of a vast experimental space became very expensive because all experiments had to be performed in a serial manner, and the chromatography systems used were designed for relatively large columns and, therefore, the experiments required large sample volumes. To address these issues, high-throughput miniaturized methods employing different operating principles and/or formats have been introduced. Herein, a technical review of the most common high-throughput formats used for the development of chromatographic purification steps is presented. The formats considered include minicolumns, prefilled pipette tips, and microtiter filter plates prefilled with chromatography resins. Advantages and limitations of each format are discussed through the prism of chromatographic theory, engineering principles, and known mass-transfer mechanisms. A roadmap for applicability of the different formats for process development purposes and implementation of a Quality by Design initiative for designing/optimization of chromatography steps is also discussed.
Method for recovery and immunoaffinity enrichment of membrane proteins illustrated with metastatic ovarian cancer tissues
Publication: International Journal of Proteomics, Vol 2012, 1-15, 2012
Integral membrane proteins play key biological roles in cell signaling, transport, and pathogen invasion. However, quantitative clinical assays for this critical class of proteins remain elusive and are generally limited to serum-soluble extracellular fragments. Furthermore, classic proteomic approaches to membrane protein analysis typically involve proteolytic digestion of the soluble pieces, resulting in separation of intra- and extracellular segments and significant informational loss. In this paper, we describe the development of a new method for the quantitative extraction of intact integral membrane proteins (including GPCRs) from solid metastatic ovarian tumors using pressure cycling technology in combination with a new (ProteoSolve-TD) buffer system. This new extraction buffer is compatible with immunoaffinity methods (e.g., ELISA and immunoaffinity chromatography), as well as conventional proteomic techniques (e.g., 2D gels, western blots). We demonstrate near quantitative recovery of membrane proteins EDG2, EDG4, FASLG, KDR, and LAMP-3 by western blots. We have also adapted commercial ELISAs for serum-soluble membrane protein fragments (e.g., sVEGFR2) to measure the tissue titers of their transmembrane progenitors. Finally, we demonstrate the compatibility of the new buffers with immunoaffinity enrichment/mass spectrometric characterization of tissue proteins.
Chip-based CE for rapid separation of 8-aminopyrene-1,3,6-trisulfonic acid (APTS) derivatized glycans
Publication: Electrophoresis, Vol 31, No 22, 3783-3786, November 2010
Fluorescently labeled carbohydrates released from glycoproteins were separated using a commercially available microfluidic chip electrophoresis system. While the instrumentation was primarily designed for DNA analysis it was found that the application base can be easily expanded using the development software provided by the manufacturer. The carbohydrates were released by enzymatic digestion (PNGase F) from glycoproteins present in human plasma after boronic acid – lectin affinity enrichment. After fluorescent labeling with 8-aminopyrene-1,3,6-trisulfonic acid the carbohydrates were separated based on capillary gel electrophoresis mechanism and detected by a fluorescence detector using a blue (470 nm) LED. The separation was completed in 40 s in a microfluidic channel of 14 mm length. Glucose ladder carbohydrate oligomers differing by one glucose unit were baseline separated up to a 20-mer with the main limitation being the detection sensitivity. As expected, the observed resolution in these experiments did not approach that of standard CE with 20 times longer separation distance; however, the chip-based analysis excelled in the speed of the separation. Similar electrophoretic profiles of glycans released from plasma glycoproteins were obtained using a standard CE equipment with 35 cm separation length and microfluidic chips with a separation distance of only 14 mm.
Mass Spectrometric-Based Stable Isotopic 2-Aminobenzoic Acid Glycan Mapping for Rapid Glycan Screening of Biotherapeutics
Publication: Analytical Chemistry, Vol 82, No 4, 1498-1508, February 2010
Fast, sensitive, robust methods for “high-level” glycan screening are necessary during various stages of a biotherapeutic product’s lifecycle, including clone selection, process changes, and quality control for lot release testing. Traditional glycan screening involves chromatographic or electrophoretic separation-based methods, and, although reproducible, these methods can be time-consuming. Even ultrahigh-performance chromatographic and microfluidic integrated LC/MS systems, which work on the tens of minute time scale, become lengthy when hundreds of samples are to be analyzed. Comparatively, a direct infusion mass spectrometry (MS)-based glycan screening method acquires data on a millisecond time scale, exhibits exquisite sensitivity and reproducibility, and is amenable to automated peak annotation. In addition, characterization of glycan species via sequential mass spectrometry can be performed simultaneously. Here, we demonstrate a quantitative high-throughput MS-based mapping approach using stable isotope 2-aminobenzoic acid (2-AA) for rapid “high-level” glycan screening.
Widening the bottleneck: increasing success in protein expression and purification
Publication: Journal of Structural Biology, Vol 172, No 1, 14-20, 2010
The number of variables at play in the expression and purification of a single protein dwarf those involved in sequencing a genome. Although certain trends are apparent, there is no one-size-fits-all approach to the process of purifying proteins. Thus, whereas numerous genome sequencing projects are providing an overwhelming number of interesting open reading frames for structural biologists to study, fully realizing the potential of this resource is still only a distant hope. We will discuss several current approaches to high throughput expression and purification as well as strategies that have served us well to quickly identify lead protein expression constructs in the context of a core service protein expression and purification laboratory. The use of the baculovirus expression vector system and implementation of a purification screening method will be emphasized.
RNA Purification and Analysis: Sample Preparation, Extraction, Chromatography
Publication: Gjerde et al., Wiley-VCH, July 2009
This first book on the market covers the many new and important RNA species discovered over the past five years, explaining current methods for the enrichment, separation and purification of these novel RNAs.
Building up from general principles of RNA biochemistry and biophysics, this book addresses the practical aspects relevant to the laboratory researcher throughout, while discussing the performance and potential problems of the methods discussed. An appendix contains a glossary with the important terms and techniques used in RNA analysis.
By explaining the basic and working principles of the methods, the book allows biochemists and molecular biologists to gain much more expertise than by simply repeating a pre-formulated protocol, enabling them to select the procedure and materials best suited to the RNA analysis task at hand. As a result, they will be able to develop new protocols where needed and optimize and fine-tune the general purpose standard protocols that come with the purification equipment and instrumentation.
Publication: Fritz et al. Wiley-VCH, February 2009
This completely revised and updated fourth edition of the best-selling classic is a thorough treatment of the subject while remaining concise and readable. New additions include capillary electrophoresis, monolithic columns, zwitterion colums, DNA/RNA analysis, fundamentals of the science of IC, and micro methods. The whole is rounded off by handy tables with details on detection or elution conditions, among others.
Fluorescent isotope-coded affinity tag 2: peptide labeling and affinity capture
Publication: Electrophoresis, Vol 30, No 7, 1111-1118, 2009
Fluorescent isotope-coded affinity tag (FCAT) is a novel reagent to label cysteine-containing peptides. The fluorescein group on the tag enables absolute quantification by fluorescence detection, also supporting affinity capture of the labeled peptides. In this paper we report the synthesis of the heavy isotopic form of the FCAT reagent and its use in labeling tryptic peptides. The heavy form of the reagent exhibited the same reactivity and chromatographic behavior that of the light version. Effective labeling of tryptic peptides from alpha-lactalbumin, fetuin, BSA and phosphorylase b was attained by using both the heavy and light FCAT tags. Selective capture of the FCAT-labeled peptides was easily performed by pipette tips containing either anti-FITC antibody or iminodiacetic-acid-coated beads. The differently labeled peptides were separated by RP HPLC and analyzed by MALDI-TOF MS.
Enrichment of Amadori products derived from the nonenzymatic glycation of proteins using microscale boronate affinity chromatography
Publication: Analytical Biochemistry, Vol 393, No 1, 8-22, 2009
Amadori peptides were enriched using boronate affinity tips and measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). As demonstrated by electrochemical measurements, the tips show the highest binding efficiency for glucose at pH 8.2 employing ammonium chloride/ammonia buffer with ionic strength of 150 mM, exceeding taurine buffer at the same concentration. The bound constituents were released by sorbitol and formic acid. It was also demonstrated that elution with sorbitol at 1.2 M is superior to acidic media. Comparison of results was based on the numbers of detected peptides and their glycated sites. Using sorbitol for elution requires desalting prior to analysis. Therefore, three different sorbents were tested: fullerene-derivatized silica, ZipTip (C18), and C18 silica. Fullerene-derivatized silica and ZipTip showed the same performance regarding the numbers of glycated peptides, and sites were better than C18 silica. The elaborated off-line method was compared with liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements, by which considerable less modified peptides were detected. Affinity tips used under optimized conditions were tested for the analysis of human serum albumin (HSA) from sera of healthy and diabetic individuals. A peptide with a mass of 1783.9 Da could be detected only in samples of diabetic patients and, therefore, could be a very interesting biomarker candidate.
High-throughput immunoglobulin G N-glycan characterization using rapid resolution reverse-phase chromatography tandem mass spectrometry
Publication: Analytical Biochemistry, Vol 385, No 1, 69-79, 2009
We present an optimized high-throughput method for the characterization of 2-aminobenzamide (2-AB)-labeled N-glycans from recombinant immunoglobulin G (rIgG). This method includes an optimized sample preparation protocol involving microwave-assisted deglycosylation in conjunction with an automated sample cleanup strategy and a rapid resolution reverse-phase high-performance liquid chromatography (RRRP-HPLC) separation of labeled N-glycans. The RRRP-HPLC method permits generation of a comprehensive glycan profile using fluorescence detection in 45min. In addition, the profiling method is directly compatible with electrospray ionization mass spectrometry (ESI-MS), allowing immediate and sensitive characterization of the glycan moiety by intact MS and tandem MS (MS/MS) fragmentation. We conservatively estimate an efficiency gain of fourfold with respect to the throughput capabilities of this optimized method as compared with traditional protocols (overnight deglycosylation, sample cleanup by graphitized carbon or cellulose cartridge, high-pH anion exchange chromatography, fraction collection, and processing for matrix-assisted laser desorption/ionization time-of-flight [MALDI-TOF] MS analysis) for a single sample. Even greater gains are achieved when processing of multiple samples is considered.
Fluorescent isotope-coded affinity tag (FCAT). I: Design and synthesis
Publication: Bioorganic Chemistry, Vol 36, No 6, 299-311, December 2008
A novel class of isotope-coded affinity tag is proposed possessing a fluorescent feature, referred to as fluorescent isotope-coded affinity tag (FCAT), to provide a new tool for quantitative proteomics. The label is designed to bind cysteine containing proteins or peptides. The FCAT reagent comprises four functional elements: a specific chemical reactivity group toward sulfhydryl groups; a linker that can incorporate the stable isotopes; a hydroxymethylbenzoic residue (base labile group) to cleave off a large part of the label before MS analysis; and a fluorescent tag for absolute quantification. The fluorescent part of the tag is also planned to be utilized to isolate the FCAT-labeled peptides via antibody based pull-down method. In this paper, we report on the solid phase organic synthesis of the light isotope containing FCAT molecule. The new labeling reagent showed good reactivity with model cysteine containing peptides. The fluorophore group was also effectively cleaved off from the labeled products to accommodate easier MS based analysis.
A simple high-throughput purification method for hit identification in protein screening
Publication: Journal of Immunological Methods, Vol 339, No 1, 38-46, November 2008
Phage and ribosome display technologies have emerged as important tools in the high-throughput screening of protein pharmaceuticals. However, a challenge created by the implementation of such tools is the need to purify large numbers of proteins for screening. While some assays may be compatible with crude bacterial lysates or periplasmic extracts, many functional assays, particularly cell-based assays, require protein of high purity and concentration. Here we evaluate several methods for small-scale, high-throughput protein purification. From our initial assessment we identified the HIS-Select 96-well filter plate system as the method of choice for further evaluation. This method was optimized and used to produce scFvs that were tested in cell-based functional assays. The behavior of HIS-Select purified scFvs in these assays was found to be similar to scFvs purified using a traditional large-scale 2-step purification method. The HIS-Select method allows high-throughput purification of hundreds of scFvs with yields in the 50-100 microg range, and of sufficient purity to allow evaluation in a cell-based proliferation assay. In addition, the use of a similar 96-well-based method facilitates the purification and subsequent screening of large numbers of IgGs and Fc fusion proteins generated through reformatting of scFv fragments.
Selection against undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podocalyxin-like protein-1
Publication: Stem Cells, Vol 26, No 6, 1454-1463, June 2008
Future therapeutic applications of differentiated human embryonic stem cells (hESC) carry a risk of teratoma formation by contaminating undifferentiated hESC. We generated 10 monoclonal antibodies (mAbs) against surface antigens of undifferentiated hESC, showing strong reactivity against undifferentiated, but not differentiated hESC. The mAbs did not cross react with mouse fibroblasts and showed weak to no reactivity against human embryonal carcinoma cells. Notably, one antibody (mAb 84) is cytotoxic to undifferentiated hESC and NCCIT cells in a concentration-dependent, complement-independent manner. mAb 84 induced cell death of undifferentiated, but not differentiated hESC within 30 minutes of incubation, and immunoprecipitation of the mAb-antigen complex revealed that the antigen is podocalyxin-like protein-1. Importantly, we observed absence of tumor formation when hESC and NCCIT cells were treated with mAb 84 prior to transplantation into severe combined immunodeficiency mice. Our data indicate that mAb 84 may be useful in eliminating residual hESC from differentiated cells populations for clinical applications. Disclosure of potential conflicts of interest is found at the end of this article.
Boronic acid lectin affinity chromatography (BLAC). 2. Affinity micropartitioning-mediated comparative glycosylation profiling
Publication: Analytical and Bioanalytical Chemistry, Vol 392, No 1-2, 195-201, 2008
As a continuation of our work on boronic acid lectin affinity chromatography (BLAC), in this paper we introduce an automated affinity micropartitioning approach using combined boronic acid and concanavalin A (BLAC/Con A) resin-filled micropipette tips to isolate and enrich human serum glycoproteins. The N-linked oligosaccharides of the partitioned glycoproteins were removed by PNGase F enzyme digestion, followed by 8-aminopyrene-1,3,6-trisulfonic acid labeling. Capillary gel electrophoresis with blue LED-induced fluorescence detection was applied in a multiplexed format for comparative glycan profiling. The efficiency of BLAC affinity micropartitioning was compared with that of the individual lectin and pseudolectin affinity enrichment. Finally, we report on our findings in glycosylation differences in human serum samples from healthy and prostate cancer patients by applying BLAC/Con A micropipette tip-based enrichment and comparative multicapillary gel electrophoresis analysis of the released and labeled glycans.
Sample preparation for the analysis of complex carbohydrates by multicapillary gel electrophoresis with light-emitting diode induced fluorescence detection
Publication: Analytical Chemistry, Vol 80, No 11, 4241-4246, 2008
This paper evaluates various sample preparation methods for multicapillary gel electrophoresis based glycan analysis to support electrokinetic injection. First the removal of excess derivatization reagent is discussed. Although the Sephadex G10 filled multiscreen 96-well filter plate and Sephadex G10 filled pipet tips enabled increased analysis sensitivity, polyamide DPA-6S pipet tips worked particularly well. In this latter case an automated liquid handling system was used to increase purification throughput, necessary to feed the multicapillary electrophoresis unit. Problems associated with the high glucose content of such biological samples as normal human plasma were solved by applying ultrafiltration. Finally, a volatile buffer system was developed for exoglycosidase-based carbohydrate analysis.
A microscale yeast cell disruption technique for integrated process development strategies
Publication: Biotechnology Progress, Vol 24, No 3, 606-614, 2008
Miniaturizing protein purification processes at the microliter scale (microscale) holds the promise of accelerating process development by enabling multi-parallel experimentation and automation. For intracellular proteins expressed in yeast, small-scale cell breakage methods capable of disrupting the rigid cell wall are needed that can match the protein release and contaminant profile of full-scale methods like homogenization, thereby enabling representative studies of subsequent downstream operations to be performed. In this study, a noncontact method known as adaptive focused acoustics (AFA) was optimized for the disruption of milligram quantities of yeast cells for the subsequent purification of recombinant human papillomavirus (HPV) virus-like particles (VLPs). AFA operates by delivering highly focused, computer-controlled acoustic radiation at frequencies significantly higher than those used in conventional sonication. With this method, the total soluble protein release was equivalent to that of laboratory-scale homogenization, and cell disruption was evident by light microscopy. The recovery of VLPs through a microscale chromatographic purification following AFA treatment was within 10% of that obtained using homogenization, with equivalent product purity. The addition of a yeast lytic enzyme prior to cell disruption reduced processing time by nearly 3-fold and further improved the comparability of the lysate to that of the laboratory-scale homogenate. In addition, unlike conventional sonication methods, sample heating was minimized (< =8 degrees C increase), even using the maximum power settings required for yeast cell disruption. This disruption technique in combination with microscale chromatographic methods for protein purification enables a strategy for the rapid process development of intracellularly expressed proteins.
Boronic acid-lectin affinity chromatography. 1. Simultaneous glycoprotein binding with selective or combined elution
Publication: Analytical and Bioanalytical Chemistry, Vol 389, No 7-8, 2097-2102, 2007
We introduce a novel combination of boronic acid affinity chromatography with lectin affinity chromatography, dubbed as boronic acid-lectin affinity chromatography (BLAC). Concanavalin A and wheat germ agglutinin lectins were mixed with the pesudo-lectin boronic acid to form the BLAC affinity column and their performance was evaluated with standard glycoproteins. Optimization of the binding and elution buffers for the BLAC system is described. The BLAC columns were employed to isolate glycoproteins of interest using both selective and/or combined elution.
Automated sample preparation facilitated by PhyNexus MEA purification system for oligosaccharide mapping of glycoproteins
Publication: Analytical Biochemistry, Vol 369, No 2, 202-209, 2007
A reproducible high-throughput sample cleanup method for fluorescent oligosaccharide mapping of glycoproteins is described. Oligosaccharides are released from glycoproteins using PNGase F and labeled with 2-aminobenzoic acid (anthranilic acid, AA). A PhyNexus MEA system was adapted for automated isolation of the fluorescently labeled oligosaccharides from the reaction mixture prior to mapping by HPLC. The oligosaccharide purification uses a normal-phase polyamide resin (DPA-6S) in custom-made pipette tips. The resin volume, wash, and elution steps involved were optimized to obtain high recovery of oligosaccharides with the least amount of contaminating free fluorescent dye in the shortest amount of time. The automated protocol for sample cleanup eliminated all manual manipulations with a recycle time of 23 min. We have reduced the amount of excess AA by 150-fold, allowing quantitative oligosaccharide mapping from as little as 500 ng digested recombinant immunoglobulin G (rIgG). This low sample requirement allows early selection of a cell line with desired characteristics (e.g., oligosaccharide profile and high specific productivity) for the production of glycoprotein drugs. In addition, the use of Tecan or another robotic platform in conjunction with this method should allow the cleanup of 96 samples in 23 min, a significant decrease in the amount of time currently required to process such a large number of samples.
An automated microscale chromatographic purification of virus-like particles as a strategy for process development
Publication: Biotechnology and Applied Biochemistry, Vol 47, No Pt 2., 131-139, 2007
The development of fermentation processes for recombinant vaccines requires optimizing expression while maintaining high product quality. Changes to cell fermentation conditions are typically evaluated following cell disruption, with expression levels quantified by immunoassay, liquid chromatography or enzyme activity. However, assay titres do not always predict the effects that intracellular aggregation, proteolysis, post-translational modifications and differences in relative impurity levels can have on purification yield and product purity. Furthermore, heterogeneity in the size and surface properties inherent in viral particles makes unit operations such as chromatography less predictable. In these cases, the purification procedure (or a mimic thereof) must be carried out to give accurate information on the impact of changes in fermentation conditions on purification process performance. This was demonstrated for the development of a recombinant vaccine against human papillomavirus produced in Saccharomyces cerevisiae, where the most informative feedback on fermentation variables was obtained by completing a multistep chromatographic purification to evaluate process yield and product purity. To increase the purification throughput and reduce labour, the chromatography was miniaturized 1000-fold from the laboratory scale using microlitre volumes of adsorbent in a pipette tip and automated on a robotic workstation. The microscale purification is shown to be predictive of the laboratory-scale purification in terms of yield and purity, while providing over a 10-fold increase in throughput and allowing for increased monitoring of fermentation processes. In addition, by reducing the volume of cells needed for this assessment, the fermentation can be correspondingly reduced in scale and carried out in parallel for additional throughput gains.
Micro-scale open-tube capillary separations of functional proteins
Publication: Analytical Biochemistry, Vol 350, No 1, 128-137, 2006
This article describes a novel technique whereby fully functional proteins or multiprotein complexes are efficiently extracted from biological samples to chemically derivatized walls of fused-silica open-tube capillary columns. Proteins are eluted with very high yields into elution volumes that are smaller in volume than the internal volume of the open-tube capillary column itself, thereby achieving 100-fold increases in target protein concentrations from starting samples of less than 1 ml. The open-tube capillary columns are designed for single use; combined with the physical and chemical characteristics of the open-tube capillary column, this provides exceptional purity to the eluted proteins. Affinity-based open-tube capillary columns are demonstrated here to purify, enrich, and maintain functionality for a monomeric and dimeric enzyme, a low-abundance HeLa nuclear complex, and a light-harvesting octadecameric membrane protein complex. The design of the open-tube capillary column allows for facile direction of the processed protein sample to any number of final detection techniques and is capable of generating final protein concentrations required for many structural biology experiments. The open-tube capillary columns are also characterized by exceptional ease of use. Current designs allow for up to 10 open-tube capillary columns to be applied simultaneously with no fundamental impediments to even greater parallel operation.
High-throughput affinity ranking of antibodies using surface plasmon resonance microarrays
Publication: Analytical Biochemistry, Vol 351, No 2, 241-253, 2006
A method was developed to rapidly identify high-affinity human antibodies from phage display library selection outputs. It combines high-throughput Fab fragment expression and purification with surface plasmon resonance (SPR) microarrays to determine kinetic constants (kon and koff) for 96 different Fab fragments in a single experiment. Fabs against human tissue kallikrein 1 (hK1, KLK1 gene product) were discovered by phage display, expressed in Escherichia coli in batches of 96, and purified using protein A PhyTip columns. Kinetic constants were obtained for 191 unique anti-hK1 Fabs using the Flexchip SPR microarray device. The highest affinity Fabs discovered had dissociation constants of less than 1 nM. The described SPR method was also used to categorize Fabs according to their ability to recognize an apparent active site epitope. The ability to rapidly determine the affinities of hundreds of antibodies significantly accelerates the discovery of high-affinity antibody leads.
Material-enhanced laser desorption/ionization (MELDI)--a new protein profiling tool utilizing specific carrier materials for time of flight mass spectrometric analysis
Publication: Journal of the American Society for Mass Spectrometry, Vol 17, No 9, 1203-1208, 2006
Over the past couple of years, proteomics pattern analysis has emerged as an effective method for the early diagnosis of diseases such as ovarian, breast, or prostate cancer, without identification of single biomarkers. MALDI-TOF MS, for example, offers a simple approach for fast and reliable protein profiling, especially by using carrier materials with various physical and chemical properties, in combination with a MALDI matrix. This approach is referred to as material-enhanced laser desorption/ionization (MELDI). In this paper, we report the development and application of derivatized carrier materials [cellulose, silica, poly(glycidyl methacrylate/divinylbenzene) (GMA/DVB) particles, and diamond powder] for fast and direct MALDI-TOF MS protein profiling. The applicability of MELDI for rapid protein profiling was evaluated with human serum samples. These carriers, having various hydrophobicities, resulted in characteristic mass fingerprints, even if all materials were derivatized with iminodiacetic acid (IDA) to yield an immobilized metal affinity chromatography (IMAC) functionality. Our study demonstrates that analyzing complex biological samples, such as human serum, by employing different MELDI carrier materials yielded type- and size-dependent performance variation.
First meeting on Developments in Protein Interaction Analysis (DiPIA 2005)
Striving for purity: advances in protein purification
Publication: Nature Methods, Vol 2, No 1, 71-77, 2005
A substantial bottleneck in working with proteins, both native and recombinant, is purifying the protein of interest efficiently, with a minimum of labor and cost. Recent advances in purification
technology from many companies are making the protein scientist’s job easier. Caitlin Smith reports.
Drug discovery – antibody enrichment and characterization
Protein purification: pure but not simple