Absolute protein quantification based on calibrated particle counting using electrospray-differential mobility analysis.

The traceability of in vitro diagnostics or drug products is based on the accurate quantification of proteins. In this study, we developed an absolute quantification approach for proteins. This method is based on calibrated particle counting using electrospray-differential mobility analysis (ES-DMA) coupled with a condensation particle counter (CPC). The absolute concentration of proteins was quantified with the observed protein particle number measured with ES-DMA-CPC, and the detection efficiency was determined by calibrators. The measurement performance and quantitative level were verified using two certificated reference materials, BSA and NIMCmAb. The linear regression fit for the detection efficiency values of three reference materials and one highly purified protein (myoglobin, BSA, NIMCmAb and fibrinogen) indicated that the detection efficiency and the particle size distribution of these proteins exhibited a linear relationship. Moreover, to explore the suitability of the detection efficiency-particle size curve for protein quantification, the concentrations of three typical proteinaceous particles, including two high molecular weight proteins (NIST reference material 8671 and D-dimer) and one protein complex (glutathione S-transferase dimer), were determined. This work suggests that this calibrated particle counting method is an efficient approach for nondestructive, rapid and accurate quantification of proteins, especially for measuring proteinaceous particles with tremendous size and without reference standards.

Development of an isotope dilution mass spectrometry assay for the quantification of insulin based on signature peptide analysis.

An isotope dilution mass spectrometry (IDMS) method that involves peptide-based protein analysis was developed to accurately quantify insulin. In this study, a signature peptide (GFFYTPK) obtained from tryptic digestion of insulin was selected as a surrogate for insulin. Then, the optimal conditions for signature peptide analysis through mass spectrometry detection and enzymatic digestion were determined. The analytical performance of this method was assessed and validated using porcine insulin-certified reference material. The linear range of the insulin calibration curve ranged from 0.05 ~ 2 mass ratios, with recoveries ranging from 96.15 to approximately 101.15%. The limit of detection was 0.19 ng/mL, and the limit of quantification was 0.63 ng/mL. The quantitative results corresponded well with a certified value that was obtained from measuring a porcine insulin reference material with amino acid-based IDMS. In addition, the target peptide GFFYTPK can be found in other species of insulin. This method was also applied for the quantification of human insulin-certified reference material. Finally, we applied the method to quantify the concentrations of simulated serum insulin. These findings suggested that this signature peptide-based IDMS approach can accurately quantify insulin levels, can assign a certified value to insulin reference materials, and has the potential to quantify serum insulin with traceable measurements.

Global Analysis of Aggregation Profiles of Three Kinds of Immuno-Oncology mAb Drug Products Using Flow Cytometry.

Accurately quantifying the protein particles in both subvisible (1-100 µm) and submicron (≤1 µm) ranges remains a prominent challenge in the development and manufacturing of protein drugs. Due to the limitation of the sensitivity, resolution, or quantification level of various measurement systems, some instruments may not provide count information, while others can only count particles in a limited size range. Moreover, the reported concentrations of protein particles commonly have significant discrepancies owing to different methodological dynamic ranges and the detection efficiency of these analytical tools. Therefore, it is extremely difficult to accurately and comparably quantify protein particles within the desired size range at one time. To develop an efficient protein aggregation measurement method that can span the entire range of interest, we established, in this study, a single particle-sizing/counting method based on our highly sensitive lab-built flow cytometry (FCM) system. The performance of this method was assessed, and its capability of identifying and counting microspheres between 0.2 and 25 µm was demonstrated. It was also used to characterize and quantify both subvisible and submicron particles in three kinds of top-selling immuno-oncology antibody drugs and their lab-produced counterparts. These assessment and measurement results suggest that there may be a role for an enhanced FCM system as an efficient investigative tool for characterizing and learning the molecular aggregation behavior, stability, or safety risk of protein products.

Global characterization of modifications to the charge isomers of IgG antibody.

Posttranslational modifications of antibody products affect their stability, charge distribution, and drug activity and are thus a critical quality attribute. The comprehensive mapping of antibody modifications and different charge isomers (CIs) is of utmost importance, but is challenging. We intended to quantitatively characterize the posttranslational modification status of CIs of antibody drugs and explore the impact of posttranslational modifications on charge heterogeneity. The CIs of antibodies were fractionated by strong cation exchange chromatography and verified by capillary isoelectric focusing-whole column imaging detection, followed by stepwise structural characterization at three levels. First, the differences between CIs were explored at the intact protein level using a top-down mass spectrometry approach; this showed differences in glycoforms and deamidation status. Second, at the peptide level, common modifications of oxidation, deamidation, and glycosylation were identified. Peptide mapping showed nonuniform deamidation and glycoform distribution among CIs. In total, 10 N-glycoforms were detected by peptide mapping. Finally, an in-depth analysis of glycan variants of CIs was performed through the detection of enriched glycopeptides. Qualitative and quantitative analyses demonstrated the dynamics of 24 N-glycoforms. The results revealed that sialic acid modification is a critical factor accounting for charge heterogeneity, which is otherwise missed in peptide mapping and intact molecular weight analyses. This study demonstrated the importance of the comprehensive analyses of antibody CIs and provides a reference method for the quality control of biopharmaceutical analysis.

Light-scattering detection within the difficult size range of protein particle measurement using flow cytometry.

The phenomenon of protein aggregation is a prominent challenge that impacts biopharmaceutical development at every stage. It may have a number of deleterious effects on protein drugs, including the loss of efficacy, induction of immunogenicity, altered pharmacokinetics and reduced shelf life. At present, multiple methods are available for counting and sizing particles over a broad range of sizes. However, there remains a conundrum in the measurement of particles in the submicrometer range, from 100 nm to 2 µm. In this study, the capability of our new laboratory built FCM system to detect model polystyrene (PS) and silica (SiO2) submicrometer microspheres was evaluated and benchmarked against flow field-flow fractionation (FFF). The FCM system showed its advantages on sensitivity, selectivity, reproducibility and speed. The laboratory-built FCM system can readily analyze model PS and SiO2 microspheres down to 200 nm, covering much of the difficult range from 100 nm to 2 µm. Our data also showed that this machine was able to monitor the distribution of antibody aggregates ranged between 200 nm and 10 µm, suggesting its usability for characterizing protein aggregation in future.

Enhancers Improve the AID-Induced Hypermutation in Episomal Vector for Antibody Affinity Maturation in Mammalian Cell Display.

The induction of somatic hypermutation (SHM) in various cell lines by activation-induced cytidine deaminase (AID) has been used in protein-directed selection, especially in antibody affinity maturation. Several antibody affinity maturation systems based on mammalian cells have been developed in recent years, i.e., 293T, H1299, Raji and CHO cells. However, the efficiency of in vitro AID-induced hypermutation is low, restricting the application of such systems. In this study, we examined the role of Ig and Ek enhancers in enhancing SHM in the episomal vector pCEP4 that expresses an anti-high mobility group box 1 (HMGB1) full-length antibody. The plasmid containing the two enhancers exhibited two-fold improvement of mutation rate over pCEP4 in an AID expression H1299 cell line (H1299-AID). With the engineered episomal vector, we improved the affinity of this antibody in H1299-AID cells by 20-fold.

Variable Threshold Method for Determining the Boundaries of Imaged Subvisible Particles.

An accurate assessment of particle characteristics and concentrations in pharmaceutical products by flow imaging requires accurate particle sizing and morphological analysis. Analysis of images begins with the definition of particle boundaries. Commonly a single threshold defines the level for a pixel in the image to be included in the detection of particles, but depending on the threshold level, this results in either missing translucent particles or oversizing of less transparent particles due to the halos and gradients in intensity near the particle boundaries. We have developed an imaging analysis algorithm that sets the threshold for a particle based on the maximum gray value of the particle. We show that this results in tighter boundaries for particles with high contrast, while conserving the number of highly translucent particles detected. The method is implemented as a plugin for FIJI, an open-source image analysis software. The method is tested for calibration beads in water and glycerol/water solutions, a suspension of microfabricated rods, and stir-stressed aggregates made from IgG. The result is that appropriate thresholds are automatically set for solutions with a range of particle properties, and that improved boundaries will allow for more accurate sizing results and potentially improved particle classification studies.

Correcting the Relative Bias of Light Obscuration and Flow Imaging Particle Counters.

PURPOSE: Industry and regulatory bodies desire more accurate methods for counting and characterizing particles. Measurements of proteinaceous-particle concentrations by light obscuration and flow imaging can differ by factors of ten or more. METHODS: We propose methods to correct the diameters reported by light obscuration and flow imaging instruments. For light obscuration, diameters were rescaled based on characterization of the refractive index of typical particles and a light scattering model for the extinction efficiency factor. The light obscuration models are applicable for either homogeneous materials (e.g., silicone oil) or for chemically homogeneous, but spatially non-uniform aggregates (e.g., protein aggregates). For flow imaging, the method relied on calibration of the instrument with silica beads suspended in water-glycerol mixtures. RESULTS: These methods were applied to a silicone-oil droplet suspension and four particle suspensions containing particles produced from heat stressed and agitated human serum albumin, agitated polyclonal immunoglobulin, and abraded ethylene tetrafluoroethylene polymer. All suspensions were measured by two flow imaging and one light obscuration apparatus. Prior to correction, results from the three instruments disagreed by a factor ranging from 3.1 to 48 in particle concentration over the size range from 2 to 20 µm. Bias corrections reduced the disagreement from an average factor of 14 down to an average factor of 1.5. CONCLUSIONS: The methods presented show promise in reducing the relative bias between light obscuration and flow imaging.

The effect of ionizing radiation on mRNA levels of the DNA damage response genes rad9, rad1 and hus1 in various mouse tissues.

Rad9, Rad1 and Hus1 are essential genes conserved from yeast to humans. They form a heterotrimer complex (9-1-1 complex) that participates in the cell cycle checkpoint activation and DNA damage repair in eukaryotic cells. Rad9, Rad1 and Hus1 deficient cells are hypersensitive to ionizing radiation and mouse cells deleted for anyone of the three genes are highly sensitive to the killing by gamma rays. We propose that ionizing radiation-induced transcription of these genes is a mechanism by which cells respond to radiation-induced damage. In this study we used quantitative real-time RT-PCR(qPCR) to analyze the mRNA levels of Rad9, Rad1 and Hus1 in various tissues isolated from mice that were either mock irradiated or exposed to 10 Gy gamma radiation. Our results indicated that the mRNA levels of Rad9, Rad1 and Hus1 genes were very different among these tissues, and we found high natural levels of mRNA in the spleen, lung, ovary and testis of mice before exposure to radiation. The mRNA levels of the three genes were well correlated across these tissues, being high, medium or low in each of the tissues simultaneously. The mRNA levels of the three genes were analyzed at 2, 6, 12, 24 and 48 h after irradiation. In most tissues Rad9 was strongly induced at 2 and 12 h time points and Hus1 was strongly induced at 2, 12 and 48 h time points, but Rad1 was minimally induced in most of the tissues with the exception of slightly higher levels in the heart and lung tissues at the 48 h time point. These results suggest that the regulation mechanisms for the mRNA levels of the three genes in response to ionizing radiation are complex and not well orchestrated. We also detected the induction of Rad9 and Hus1 proteins in the heart and liver of the animals after irradiation, and found that Rad9 protein levels were highly induced in both the heart and liver, while the Hus1 protein levels were significantly induced only in the liver, suggesting that Rad9 and Hus1 protein levels are not regulated in a coordinated manner in response to irradiation. We then went on to measure the mRNA levels of the three genes and the Rad9 and Hus1 protein levels in the mouse liver cell line (NCTC 1469) in response to irradiation in vitro. All three genes in the cultured cells were minimally induced at mRNA level, obviously different from the highly dynamic induction in liver. Rad9 and Hus1 were significantly induced at the protein level, but the induced Rad9 protein levels were higher than the Hus1 levels. Taken together, the good correlation of the mRNA levels of Rad9, Hus1 and Rad1 genes across different tissues isolated from the animals that were mock irradiated and the lack of correlation in mRNA as well as protein levels after irradiation suggest that the 9-1-1 complex has evolved to play various physiological roles in tissues rather than dealing with high doses of gamma radiation or other genotoxic agents.

Particle shape effects on subvisible particle sizing measurements.

Particle analysis tools for the subvisible (<100 µm) size range, such as light obscuration, flow imaging (FI), and electrical sensing zone (ESZ), often produce results that do not agree with one another, despite their general agreement when characterizing polystyrene latex spheres of different sizes. To include the effect of shape in comparison studies, we have used the methods of photolithography to create rods and disks. Although the rods are highly monodisperse, the instruments produce broadened peaks and report mean size parameters that are different for different instruments. We have fabricated a microfluidic device that simultaneously performs ESZ and FI measurements on each particle to elucidate the causes of discrepancies and broadening. Alignment of the rods with flow causes an oversizing by FI and undersizing by ESZ. FI also oversizes rods because of the incorrect edge definition that results from diffraction and imperfect focus. We present an improved correction algorithm for this effect that reduces discrepancies for rod-shaped particles. Tumbling of particles is observed in the microfluidic ESZ/FI and results in particle oversizing and breadth of size distribution for the monodisperse rods.

An interlaboratory comparison of sizing and counting of subvisible particles mimicking protein aggregates.

Accurate counting and sizing of protein particles has been limited by discrepancies of counts obtained by different methods. To understand the bias and repeatability of techniques in common use in the biopharmaceutical community, the National Institute of Standards and Technology has conducted an interlaboratory comparison for sizing and counting subvisible particles from 1 to 25 µm. Twenty-three laboratories from industry, government, and academic institutions participated. The circulated samples consisted of a polydisperse suspension of abraded ethylene tetrafluoroethylene particles, which closely mimic the optical contrast and morphology of protein particles. For restricted data sets, agreement between data sets was reasonably good: relative standard deviations (RSDs) of approximately 25% for light obscuration counts with lower diameter limits from 1 to 5 µm, and approximately 30% for flow imaging with specified manufacturer and instrument setting. RSDs of the reported counts for unrestricted data sets were approximately 50% for both light obscuration and flow imaging. Differences between instrument manufacturers were not statistically significant for light obscuration but were significant for flow imaging. We also report a method for accounting for differences in the reported diameter for flow imaging and electrical sensing zone techniques; the method worked well for diameters greater than 15 µm.

The Use of Index-Matched Beads in Optical Particle Counters.

In this paper, we demonstrate the use of 2-pyridinemethanol (2P) aqueous solutions as a refractive index matching liquid. The high refractive index and low viscosity of 2P-water mixtures enables refractive index matching of beads that cannot be index matched with glycerol-water or sucrose-water solutions, such as silica beads that have the refractive index of bulk fused silica or of polymethylmethacrylate beads. Suspensions of beads in a nearly index-matching liquid are a useful tool to understand the response of particle counting instruments to particles of low optical contrast, such as aggregated protein particles. Data from flow imaging and light obscuration instruments are presented for bead diameters ranging from 6 µm to 69 µm, in a matrix liquid spanning the point of matched refractive index.

Targeted deletion of mouse Rad1 leads to deficient cellular DNA damage responses.

The Rad1 gene is evolutionarily conserved from yeast to human. The fission yeast Schizosaccharomyces pombe Rad1 ortholog promotes cell survival against DNA damage and is required for G(2)/M checkpoint activation. In this study, mouse embryonic stem (ES) cells with a targeted deletion of Mrad1, the mouse ortholog of this gene, were created to evaluate its function in mammalian cells. Mrad1 (-/-) ES cells were highly sensitive to ultraviolet-light (UV light), hydroxyurea (HU) and gamma rays, and were defective in G(2)/M as well as S/M checkpoints. These data indicate that Mrad1 is required for repairing DNA lesions induced by UV-light, HU and gamma rays, and for mediating G(2)/M and S/M checkpoint controls. We further demonstrated that Mrad1 plays an important role in homologous recombination repair (HRR) in ES cells, but a minor HRR role in differentiated mouse cells.

Isolation and identification of a distinct side population cancer cells in the human epidermal squamous cancer cell line A431.

Side population (SP) cells have been suggested to be multipotent cancer stem cells. To address whether SP cells exist in epidermal squamous cancer cell line A431, A431 cells dyed with Hoechst 33342 were sorted through flow cytometry. The SP cells were then analyzed by colony-forming and cell proliferation assay. Further, tumorigenicity and microarray analysis were used to compare biological difference between SP and non-SP (NSP) cells. Our results showed that SP cells existed in the A431 cell line, showing higher proliferating and colony-forming ability than NSP cells. Tumors generated from SP cells were larger than those from the NSP cells in NOD/SCID mice. The mRNA microarray profiling revealed that five cancer marker gene expressions were up-regulated and one tumor suppressor gene expression was down-regulated. These findings suggest that SP cells in A431 could contribute to self-renewal, neoplastic transformation, and cancer metastasis of human epidermal squamous cell carcinoma.

Rad9 is required for B cell proliferation and immunoglobulin class switch recombination.

B cell maturation and B cell-mediated antibody response require programmed DNA modifications such as the V(D)J recombination, the immunoglobulin (Ig) class switch recombination, and the somatic hypermutation to generate functional Igs. Many protein factors involved in DNA damage repair have been shown to be critical for the maturation and activation of B cells. Rad9 plays an important role in both DNA repair and cell cycle checkpoint control. However, its role in Ig generation has not been reported. In this study, we generated a conditional knock-out mouse line in which Rad9 is deleted specifically in B cells and investigated the function of Rad9 in B cells. The Rad9(-/-) B cells isolated from the conditional knock-out mice displayed impaired growth response and enhanced DNA lesions. Impaired Ig production in response to immunization in Rad9(-/-) mice was also detected. In addition, the Ig class switch recombination is deficient in Rad9(-/-) B cells. Taken together, Rad9 plays dual roles in generating functional antibodies and in maintaining the integrity of the whole genome in B cells.

Hydroxyurea-induced global transcriptional suppression in mouse ES cells.

Hydroxyurea (HU), as a therapeutic medicine, has been extensively used clinically. To further survey molecular mechanisms of HU treatment, we analyzed global transcriptomic alteration of mouse ES cells in response to the treatment using high-throughput sequencing. We show that the global transcriptional activity is significantly suppressed as cells are exposed to HU treatment and alters multiple key cellular pathways, including cell cycle, apoptosis and DNAs. HU treatment also alters alternative splicing mechanisms and suppresses non-coding RNA expression. Our result provides novel clues for the understanding of how cells respond to HU and further suggests that high-throughput sequencing technology provides a powerful tool to study mechanisms of clinical drugs at the cellular level.

Mouse Rad1 deletion enhances susceptibility for skin tumor development.

BACKGROUND: Cells are constantly exposed to stresses from cellular metabolites as well as environmental genotoxins. DNA damage caused by these genotoxins can be efficiently fixed by DNA repair in cooperation with cell cycle checkpoints. Unrepaired DNA lesions can lead to cell death, gene mutation and cancer. The Rad1 protein, evolutionarily conserved from yeast to humans, exists in cells as monomer as well as a component in the 9-1-1 protein complex. Rad1 plays crucial roles in DNA repair and cell cycle checkpoint control, but its contribution to carcinogenesis is unknown. RESULTS: To address this question, we constructed mice with a deletion of Mrad1. Matings between heterozygous Mrad1 mutant mice produced Mrad1+/+ and Mrad1+/- but no Mrad1-/- progeny, suggesting the Mrad1 null is embryonic lethal. Mrad1+/- mice demonstrated no overt abnormalities up to one and half years of age. DMBA-TPA combinational treatment was used to induce tumors on mouse skin. Tumors were larger, more numerous, and appeared earlier on the skin of Mrad1+/- mice compared to Mrad1+/+ animals. Keratinocytes isolated from Mrad1+/- mice had significantly more spontaneous DNA double strand breaks, proliferated slower and had slightly enhanced spontaneous apoptosis than Mrad1+/+ control cells. CONCLUSION: These data suggest that Mrad1 is important for preventing tumor development, probably through maintaining genomic integrity. The effects of heterozygous deletion of Mrad1 on proliferation and apoptosis of keratinocytes is different from those resulted from Mrad9 heterozygous deletion (from our previous study), suggesting that Mrad1 also functions independent of Mrad9 besides its role in the Mrad9-Mrad1-Mhus1 complex in mouse cells.

Rad9 plays an important role in DNA mismatch repair through physical interaction with MLH1.

Rad9 is conserved from yeast to humans and plays roles in DNA repair (homologous recombination repair, and base-pair excision repair) and cell cycle checkpoint controls. It has not previously been reported whether Rad9 is involved in DNA mismatch repair (MMR). In this study, we have demonstrated that both human and mouse Rad9 interacts physically with the MMR protein MLH1. Disruption of the interaction by a single-point mutation in Rad9 leads to significantly reduced MMR activity. This disruption does not affect S/M checkpoint control and the first round of G(2)/M checkpoint control, nor does it alter cell sensitivity to UV light, gamma rays or hydroxyurea. Our data indicate that Rad9 is an important factor in MMR and carries out its MMR function specifically through interaction with MLH1.

Targeted deletion of Rad9 in mouse skin keratinocytes enhances genotoxin-induced tumor development.

The Rad9 gene is evolutionarily conserved from yeast to humans and plays crucial roles in genomic maintenance, DNA repair, and cell cycle checkpoint controls. However, the function of this gene with respect to tumorigenesis is not well-understood. A Rad9-null mutation in mice causes embryonic lethality. In this study, we created mice in which mouse Rad9, Mrad9, was deleted only in keratinocytes to permit examination of the potential function of the gene in tumor development. Mice with Mrad9(+/-) or Mrad9(-/-) keratinocytes showed no overt, spontaneous morphologic defects and seemed similar to wild-type controls. Painting the carcinogen 7,12-dimethylbenzanthracene (DMBA) onto the skin of the animals caused earlier onset and more frequent formation of tumors and senile skin plaques in Mrad9(-/-) mice, compared with Mrad9(+/-) and Mrad9(+/+) littermates. DNA damage response genes p21, p53, and Mrad9B were expressed at higher levels in Mrad9(-/-) relative to Mrad9(+/+) skin. Keratinocytes isolated from Mrad9(-/-) skin had more spontaneous and DMBA-induced DNA double strand breaks than Mrad9(+/+) keratinocytes, and the levels were reduced by incubation with the antioxidant epigallocatechin gallate. These data suggest that Mrad9 plays an important role in maintaining genomic stability and preventing tumor development in keratinocytes.

The up-regulation of 14-3-3 proteins in Smad4 deficient epidermis and hair follicles at catagen.

Each postnatal hair follicle (HF) perpetually goes through three phases: anagen, catagen, and telogen. The molecular signals that orchestrate the follicular transition between phases are still largely unknown. Our previous study shows that the keratinocyte specific Smad4 knockout mice exhibit progressive alopecia due to the mutant HFs failure to undergo programmed regression. To investigate the detailed molecular events controlling this process, the protein profiles of Smad4 mutant and control epidermal and HF keratinocytes were compared using 2-D difference gel electrophoresis (2-D DIGE) proteomic analysis. Eighty-six differentially expressed protein spots were identified by MALDI-TOF/TOF MS or ESI-MS/MS as 72 proteins, of which 29 proteins were found to be changed during the anagen-catagen transition of HFs in Smad4 mutants compared with the controls. The differentially expressed proteins represent a wide spectrum of functional classes such as keratin, the cytoskeleton, cellular growth and differentiation, ion combination and transfer, protein enzymes. Notably, we found that the 14-3-3sigma protein together with the 14-3-3zeta and 14-3-3beta proteins were significantly down-regulated only in wild-type keratinocytes but not in Smad4 mutant keratinocytes during the catagen phase, suggesting that increased expression of 14-3-3 proteins might contribute to the blockade of catagen initiation in Smad4 deficient HFs.