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According to the laboratory revised criteria the anti-cardiolipin (ACL) IgG, IgM with purpura, ulcers in her lower extremities and splinter hemorrhages of the nails. In a bone marrow biopsy showed 15% plasma cells positive for IgG​.


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Search results for “splinter cell conviction igg”. Everything · SoundCloud Go+ tracks · Tracks · People · Albums · Playlists · Legal ⁃ Privacy ⁃ Cookies ⁃ Imprint​.


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with fibroid necrosis, extravasation of red blood cells, and interstitial oedema (​Dongre et al. PAN-like lesions, livedo racemosa, splinter haemorrhages, palmar erythema, DIF will reveal vascular immunoglobulins, predominantly IgM​, and.


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Shortages of red blood cells, white blood cells, and blood platelets are abnormal antibody (IgG. is used to remove a tiny splinter of bone and marrow.


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The first had elevated titres of aGL (IgG isotype) and suffered recurrent DVT and The second patient presented with extensive bruising, purpura and splinter Hairy-cell leukaemia in a patient with positive LA which resolved followed.


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are considered type 2 or type 3 because they are composed of both IgG and IgM,​63 and may also result in ulcerations, gangrene of distal extremities, splinter hemorrhages, Sickle Cell Disease Ulcers Sickle cell disease (SCD) was first.


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IgE immunoglobulin: Antibodies produced by the immune system. These antibodies travel to cells that release chemicals, causing an allergic reaction. such as toxins, chemicals, drugs, and foreign particles (such as a splinter) can also be.


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Anti-β2 -glycoprotein I antibodies, IgG or IgM [>99th percentile as measured by a interference with production and release of prostacyclin by endothelial cells fold ulcers • Splinter hemorrhages • Widespread cutaneous necrosis, typically as​.


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Figure 4b , c shows the subunit mass analysis of IdeS-digested shoulder fraction. Heavy chain extensions produced by splicing require transcript read through the downstream termination signal sequence, producing an extended transcript that is then spliced. Mutations at the receptor binding sequence have been reported to have various degrees of positive or negative effects on receptor binding affinity.{/INSERTKEYS}{/PARAGRAPH} This study emphasizes the power of integrating product physicochemical characterization data with cell line omics data to understand therapeutic protein sequence variants and to define screening strategies for cell lines with improved product quality profiles. For example, a point mutation in a single cell may lead to a sub-population of the production cell line that expresses a single nucleotide variant SNV , resulting in a percentage of the final drug product carrying a single amino acid substitution. In addition, the non-reducing peptide mapping analysis confirmed the cysteine adduct and one additional disulfide bond in the extension peptide. The peak at The other new peak at This mass cannot be assigned to any mAb-A peptide fragment. Unlike a single amino acid substitution, this type of sequence variant was challenging to identify from MS analysis alone and required the insights provided by the additional data from HTS to infer the variant sequence. Furthermore, nanopore long-read genomic sequencing highlighted that the aberrant fusion transcript originated from cryptic splicing of a transcript derived from an unexpected partially deleted copy of the plasmid. Quantification analysis identified the fusion transcript to be 0. This produced an extra amino acids at the C-terminus of the heavy chain, as shown in Supplementary Figure S2a,b, in agreement with the Da mass increase detected by reducing intact mass analysis. Sequence variants produced by either genomic mutations or misincorporation during translation have been reported for therapeutic protein products. The deconvoluted mass showed the front shoulder peak contained a mass Da higher than the monomer Supplementary Figure S1. There was no evidence of a recombination event where the heavy and light chain sequences found in the fusion transcript were found adjacent to each other in the encoding DNA. Intact mass analysis of the enriched shoulder peak provided an accurate mass of the variant and revealed that the additional 11 kDa molecular mass was part of the Fc domain. Advances in cell engineering methods, using site-specific recombinases 35 or CRISPR-Cas9 systems, 36 have enabled targeted integration of a defined number of copies of an expression plasmid with more predictable structures in the host genome, thereby avoiding the risk of introducing fusions from integrating partial copies of the expression plasmid or from the interface with the host genome. The presence of the complete light constant domain coding sequence at the end of the heavy chain in a fusion transcript was confirmed by RT-PCR sequence analysis. This mass matched the detected mass of the unknown peak in the shoulder fraction Figures 5 — 7a , b. The UV chromatograms of non-reducing Lys-C peptide mapping with all peaks related to the 11 kDa extension labeled are shown in Supplementary Figure S4, and the peptides listed in Table S1. Recombinant cell lines constructed by random integration can contain multiple copies of the plasmid, and, during the plasmid integration process or subsequent cell division, copies of the plasmid can be rearranged or partially deleted. C-terminal heavy chain extensions using the same cryptic donor site at the end of the CH3 domain have been reported by others. Even though low-level sequence variants can be enriched by chromatography approaches, such as size exclusion, 5 , 15 ion exchange, 21 or reversed-phase 20 chromatography, it is still time-consuming and resource-intensive to enrich enough material for multi-enzyme de novo sequencing. To decipher the sequence, the transcriptome of the manufacturing cell line was characterized by Illumina RNA-seq. Reducing Lys-C peptide mapping was performed to identify the peptide sequence corresponding to the 11 kDa variant. These non-coding components can be included in the transgene structure to increase the level of protein expression. Although inclusion of introns in a transgene increases its expression level, at the same time the pre-mRNA splicing process is complex and not entirely predictable. The amino acid translation is shown above. This new fusion transcript, and the canonical mAb-A heavy chain and light chain were added to the CHO-K1 transcriptome, and the expression level of the heavy chain fusion transcript was found to be 0. It has been reported that extra light chain may be added to the intact mAb through disulfide bonds, leading to a size variant. Therapeutic glycoproteins such as monoclonal antibodies mAbs are produced in mammalian cell production platforms, such as those based on Chinese hamster ovary CHO cell lines, which inherently generate product heterogeneity. This result suggested that the 11 kDa variant may contain extra light chain sequence. Therefore, more sensitive and data-rich HTS characterization was used to analyze both the transcriptome and the genome of the manufacturing cell line. Analysis using PCR on genomic DNA confirmed the presence of the incomplete copy of the expression vector, but no evidence of a smaller product that would be present if the fusion sequence was found in the genomic DNA, confirming the fusion transcript was produced from mis-splicing. Nucleotide sequencing technologies offer a complementary approach to identify variants encoded in genes or mature transcripts. In comparison, the front shoulder fraction contained an additional species with mass of Da, which is Da higher than the expected monomer mass Figure 2d. The difference in mass addition measured under non-reducing and reducing conditions is Da Da — Da , which is consistent with the loss of a cysteine adduct and opening of a disulfide bond after reduction. The lack of peak identification and annotation is a limiting factor for proteomics experiments that can be overcome by proteogenomics, a new field that is based on the use of high-throughput data from different sources as part of an iterative refinement process of gene models. Inset is zoomed view. However, in one situation a partial copy of the plasmid had integrated, leading to the light chain constant sequence being closer to the end of the heavy chain than would occur if the whole plasmid copy had been integrated Figure 6b. We demonstrated that analyzing the transcriptome, and understanding the integrated plasmid structure and consequent transcript cryptic splicing events were complementary to LC-MS and essential for sequence variant identification and confirmation. When protein sequence variants are present at a relatively high level, they can be detected by intact mass analysis or peptide mapping UV profile comparison. Further characterization indicated that the 11 kDa was added to the heavy chain HC Fc domain. Similarly, a variant peak migrating later than the heavy chain peak at 22 min was observed by reducing CE-SDS Figure 3b — d. Peaks at In non-reducing intact mass analysis, the monomer fraction only contained the expected monomer mass Da Figure 2a. A splice site prediction tool 31 identified a possible cryptic donor site at the end of the heavy chain. Consistent with the intact mass analysis, the shoulder fraction contained a major size variant peak that migrated later than the intact monomer peak in non-reducing CE-SDS at 30 min Figure 3a — c. For mAb therapeutics, the impact of a sequence variant on potency, pharmacokinetics PK and patient safety largely depends on the location, the nature of the variant and the mechanism of action of the therapeutic. Intact mass analysis: Deconvoluted masses for HPSEC monomer fraction a : intact mass b : reduced light chain c : reduced heavy chain and shoulder fraction d : intact mass e : reduced light chain f : reduced heavy chain. Our study demonstrates that combining protein physicochemical characterization with genomic and transcriptomic analysis of the manufacturing cell line greatly improves the identification of sequence variants and understanding of the underlying molecular mechanisms. A complementary approach was thus used to investigate variants encoded at the nucleic acid level by the manufacturing cell line. The fusion transcript could have been produced from a genomic recombination event, splicing of the RNA, or a combination of both. Identifying and characterizing any sequence variants present is essential for product development. Silent mutations, in which the mutated DNA encodes the same original amino acid, do not directly alter the protein sequence, but they can affect transcript stability, protein folding, 10 , 11 and mRNA splicing. Two new peaks were observed in the shoulder fraction. Mis-splicing causes the sequence highlighted in blue to be spliced out. Theoretically, a variant with an additional 11 kDa mass on both heavy chains could also be present. The sequence of the protein product of the fusion transcript was inferred by extending and translating its sequence up to the end of the light chain constant domain. This genomic structure provides an explanation of the heavy chain extension variant originating from cryptic splicing of an extended heavy chain transcript reading through into the downstream light chain constant domain. In particular, the de novo reconstruction analysis of the transcriptome revealed a fusion transcript encoding the variant, highlighting the high sensitivity of this method. Translation of this fusion transcript generated an extended peptide sequence at the HC C-terminus corresponding to the observed 11 kDa mass addition. The sequence of the heavy chain-light chain constant domain fusion transcript was confirmed by RT-PCR sequencing using a cDNA template derived from the manufacturing cell line with primers that bind to the CH3 domain of the heavy chain, and the polyA sequence of the light chain. Splice variants are identified only in the sequence of the mature transcript, with no trace in the corresponding genomic sequence, and therefore are more challenging to detect. Therefore, we opted for HTS genome and transcriptome analysis of the manufacturing cell line as an approach to decipher RNA and DNA sequences that could encode the variant. Despite the relatively large mass addition, only one unknown peptide was detected by peptide mapping. It is possible that the strong promoter used in the heavy chain expression cassette may increase the frequency of transcripts reading through beyond the termination signal sequences. Variants can be introduced into the encoding gene sequences during vector propagation in bacteria prior to transfection or may result from errors in DNA replication in the production cell line once the vector has been incorporated. The tandem mass analysis of this peak further verified the peptide sequence Figure 7c. Extension products could be prevented by either increasing the level of transcript termination 37 or by reducing mis-splicing. In addition, when peak heights were compared, the two peptides that had significant higher intensity in the shoulder fraction peak a and b in Figure 5 were both from the light chain constant region. Figure 5 compares the peptide map UV profiles of the shoulder and monomer fractions. It is also notable that in our study, the proposed mis-splicing event was due to the integration of a partial copy of the expression vector, which, for this reason, was not detectable by using a splice site prediction tool on the expression vector sequence and, hence, more challenging to identify. Therefore, the intact mass analysis results indicated that the product variant had Da added to the heavy chain and contained a disulfide bond linked cysteine adduct. In reducing intact mass analysis, the monomer and shoulder fractions contained the same mass for the light chain Figure 2b —e , whereas an additional species with a mass of Da was observed in the shoulder fraction for the heavy chain Figure 2f , which is Da higher than expected Figure 2c. Engineering of the DNA to remove the cryptic donor site at the end of the CH3 domain whilst maintaining the amino acid sequence was reported by Spahr et al. Hence, a proteomic approach such as multi-enzyme digestion is essential for de novo sequencing analyses. The molecular mass of the translated light chain constant domain sequence matched the extra 11 kDa detected for the Fc extension variant. Our data showed that the 11 kDa variant is covalently linked to the heavy chain Fc domain. Larger-scale genomic recombination events can produce extension products, including molecules in which entire domains were added. The fusion transcript sequence after splicing has occurred is shown covering the area of the fusion site as identified in the RNASeq data. The upper track reports two of the plasmid copies integrated into the host genome. {PARAGRAPH}{INSERTKEYS}Protein primary structure is a potential critical quality attribute for biotherapeutics. However, standard Sanger sequencing of RT-PCR products using primers to amplify the entire heavy chain transcript did not detect any sequence variants. Combining HTS of the producing cell line with MS and amino acid analyses of the product ensures comprehensive screening for sequence variants. Due to the size of the variant 11 kDa larger , de novo sequencing using peptide mapping to further interrogate the sequence variant would require a large quantity of enriched material, as well as multiple digestions with different enzymes. During early process development and product characterization, mAb-A showed a front shoulder on the high-performance size-exclusion chromatography HPSEC main peak Figure 1a. Intact mass analysis and peptide mapping were used to deduce that the 11 kDa increase in molecular mass resulted from an addition to the heavy chain Fc. Pharmaceutical drug product characterization is a regulatory requirement that ensures consistent drug product quality between manufacturing batches. The protein physicochemical analysis described above did not unambiguously elucidate the structure of the sequence variant. To verify this finding, an orthogonal size variant characterization method, CE-SDS, was used to analyze the fractions. If the 11 kDa was an extension of the C-terminus of the heavy chain, caused by a mutation in the stop codon, the levels of peptide containing this lysine residue would be much higher in the HPSEC shoulder fraction. Additionally, a simple mutation in the stop codon can also be ruled out because the peptide containing the heavy chain C-terminal lysine is barely detected in either fraction. Genomic sequencing using the MinION indicated the integration of a partial copy of the expression vector leading to the unexpected positioning of a light chain constant domain downstream of a copy of the heavy chain gene. In particular, high-throughput sequencing HTS is a powerful tool able to overcome the limitation of sensitivity typical of the traditional Sanger sequencing of reverse transcription-polymerase chain reaction RT-PCR product variants. Together these results confirmed that the extra 11 kDa mass is an addition to one of the heavy chains. Primary structure variants can also be caused by mis-splicing of introns present in the transgene. Inspecting the genomic sequencing data provided by the MinION nanopore sequencer showed that multiple copies of the plasmid had integrated into the genome in tandem.