The serological hallmark of primary biliary cirrhosis (PBC) is the presence of pyruvate dehydrogenase complex E2 subunit (PDC-E2) antimitochondrial antibodies (AMAs). Anti- PDC-E2 antibodies cross-react specifically w...The serological hallmark of primary biliary cirrhosis (PBC) is the presence of pyruvate dehydrogenase complex E2 subunit (PDC-E2) antimitochondrial antibodies (AMAs). Anti- PDC-E2 antibodies cross-react specifically with mycobacterial hsp65, and we have demonstrated that the motif SxGDL[ILV]- AE shared by PDC- E2212- 226 and hsp’s is a cross-reactive target. Having found that this same motif is present only in β -galactosidase of Lactobacillus delbrueckii (BGAL LACDE), we hypothesized that this homology would also lead to cross-reactivity. The mimics were tested via ELISA for reactivity and competitive cross-reactivity using sera from100 AMA-positive and 23 AMA-negative PBC patients and 190 controls. An Escherichia colt (ECOLI) PDC-E2 mimic that has been pathogenetically linked to PBC but lacks this motif has been also tested. Anti-BGAL266-280 LACDE antibodies were restricted to AMA-positive patients (54 of 95, 57% ) and belonged to immunoglobulin (Ig) G3. Of the 190 controls, 22 (12% ; P < .001) had anti-BGAL266-280 antibodies, mainly of the IgG4 subclass. ECOLI PDC-E2 reactivity was virtually absent. BGAL266- 280/PDC- E2212- 226 reactivity of the IgG3 isotype was found in 52 (52% ) AMA-positive PBC patients but in only 1 of the controls (P < .001). LACDE BGAL266- 280/PDCXE2212- 226 reactivity was due to cross-reactivity as confirmed via competition ELISA.Antibody affinity for BGAL266- 280 was greater than for PDC- E2 mimics. Preincubation of a multireactive serum with BGAL266- 280 reduced the inhibition of enzymatic activity by 40% , while marginal effect (12% ) or no effect (2% ) was observed in human or ECOLIPDC-E2 mimics. In conclusion, IgG3 antibodies to BGAL LACDE cross-react with the major mitochondrial autoepitope and are characteristic of PBC.展开更多
We describe a patient with widespread skin lesions and circulating IgG autoantibodies to both type VII collagen and laminin 5. Although autoantibodies to type VII collagen belonged to IgG2, IgG3, and IgG4 subclasses, ...We describe a patient with widespread skin lesions and circulating IgG autoantibodies to both type VII collagen and laminin 5. Although autoantibodies to type VII collagen belonged to IgG2, IgG3, and IgG4 subclasses, laminin 5 was targeted exclusively by IgG3 autoantibodies. Interestingly, despite the presence of IgG3 autoantibodies, the patient’ s serum failed to fix complement to the dermoepidermal junction. In addition, these autoantibodies did not recruit and activate leukocytes or induce dermoepidermal separation in skin sectioned by cryostat. We report a most unusual case of an autoimmune subepidermal blistering with an exclusive IgG3 reactivity to laminin 5.展开更多
Compared to small molecule process analytical technology （PAT） applications, biotechnology product PAT applications have certain unique challenges and opportunities. Understanding process dynamics of bioreactor cell...Compared to small molecule process analytical technology （PAT） applications, biotechnology product PAT applications have certain unique challenges and opportunities. Understanding process dynamics of bioreactor cell culture process is essential to establish an appropriate process control strategy for biotechnology product PAT applications. Inline spectroscopic techniques for real time monitoring of bioreactor cell culture process have the distinct potential to develop PAT approaches in manufac- turing biotechnology drug products. However, the use of inline Fourier transform infrared （FTIR） spectroscopic techniques for bioreactor cell culture process monitoring has not been reported. In this work, real time inline FTIR Spectroscopy was applied to a lab scale bioreactor mAb IgG3 cell culture fluid biomolecular dynamic model. The technical feasibility of using FTIR Spectroscopy for real time tracking and monitoring four key cell culture metabolites （including glucose, glutamine, lactate, and ammonia） and protein yield at increasing levels of complexity （simple binary system, fully formulated media, actual bioreactor cell culture process） was evaluated via a stepwise approach. The FTIR fingerprints of the key metabolites were identified. The multivariate partial least squares （PLS） calibration models were established to correlate the process FTIR spectra with the concentrations of key metabolites and protein yield of in-process samples, either individually for each metabolite and protein or globally for all four metabolites simultaneously. Applying the 2＇ld derivative pre-processing algorithm to the FTIR spectra helps to reduce the number of PLS latent variables needed significantly and thus simplify the interpretation of the PLS models. The validated PLS models show promise in predicting the concentration profiles of glucose, glutamine, lactate, and ammonia and protein yield over the course of the bioreactor cell culture process. Therefore, this work demonstrated the technical feasibility of real time monitoring of the bioreactor cell culture process via FTIR spectroscopy. Its implications for enabling cell culture PAT were discussed.展开更多
文摘The serological hallmark of primary biliary cirrhosis (PBC) is the presence of pyruvate dehydrogenase complex E2 subunit (PDC-E2) antimitochondrial antibodies (AMAs). Anti- PDC-E2 antibodies cross-react specifically with mycobacterial hsp65, and we have demonstrated that the motif SxGDL[ILV]- AE shared by PDC- E2212- 226 and hsp’s is a cross-reactive target. Having found that this same motif is present only in β -galactosidase of Lactobacillus delbrueckii (BGAL LACDE), we hypothesized that this homology would also lead to cross-reactivity. The mimics were tested via ELISA for reactivity and competitive cross-reactivity using sera from100 AMA-positive and 23 AMA-negative PBC patients and 190 controls. An Escherichia colt (ECOLI) PDC-E2 mimic that has been pathogenetically linked to PBC but lacks this motif has been also tested. Anti-BGAL266-280 LACDE antibodies were restricted to AMA-positive patients (54 of 95, 57% ) and belonged to immunoglobulin (Ig) G3. Of the 190 controls, 22 (12% ; P < .001) had anti-BGAL266-280 antibodies, mainly of the IgG4 subclass. ECOLI PDC-E2 reactivity was virtually absent. BGAL266- 280/PDC- E2212- 226 reactivity of the IgG3 isotype was found in 52 (52% ) AMA-positive PBC patients but in only 1 of the controls (P < .001). LACDE BGAL266- 280/PDCXE2212- 226 reactivity was due to cross-reactivity as confirmed via competition ELISA.Antibody affinity for BGAL266- 280 was greater than for PDC- E2 mimics. Preincubation of a multireactive serum with BGAL266- 280 reduced the inhibition of enzymatic activity by 40% , while marginal effect (12% ) or no effect (2% ) was observed in human or ECOLIPDC-E2 mimics. In conclusion, IgG3 antibodies to BGAL LACDE cross-react with the major mitochondrial autoepitope and are characteristic of PBC.
文摘We describe a patient with widespread skin lesions and circulating IgG autoantibodies to both type VII collagen and laminin 5. Although autoantibodies to type VII collagen belonged to IgG2, IgG3, and IgG4 subclasses, laminin 5 was targeted exclusively by IgG3 autoantibodies. Interestingly, despite the presence of IgG3 autoantibodies, the patient’ s serum failed to fix complement to the dermoepidermal junction. In addition, these autoantibodies did not recruit and activate leukocytes or induce dermoepidermal separation in skin sectioned by cryostat. We report a most unusual case of an autoimmune subepidermal blistering with an exclusive IgG3 reactivity to laminin 5.
文摘Compared to small molecule process analytical technology （PAT） applications, biotechnology product PAT applications have certain unique challenges and opportunities. Understanding process dynamics of bioreactor cell culture process is essential to establish an appropriate process control strategy for biotechnology product PAT applications. Inline spectroscopic techniques for real time monitoring of bioreactor cell culture process have the distinct potential to develop PAT approaches in manufac- turing biotechnology drug products. However, the use of inline Fourier transform infrared （FTIR） spectroscopic techniques for bioreactor cell culture process monitoring has not been reported. In this work, real time inline FTIR Spectroscopy was applied to a lab scale bioreactor mAb IgG3 cell culture fluid biomolecular dynamic model. The technical feasibility of using FTIR Spectroscopy for real time tracking and monitoring four key cell culture metabolites （including glucose, glutamine, lactate, and ammonia） and protein yield at increasing levels of complexity （simple binary system, fully formulated media, actual bioreactor cell culture process） was evaluated via a stepwise approach. The FTIR fingerprints of the key metabolites were identified. The multivariate partial least squares （PLS） calibration models were established to correlate the process FTIR spectra with the concentrations of key metabolites and protein yield of in-process samples, either individually for each metabolite and protein or globally for all four metabolites simultaneously. Applying the 2＇ld derivative pre-processing algorithm to the FTIR spectra helps to reduce the number of PLS latent variables needed significantly and thus simplify the interpretation of the PLS models. The validated PLS models show promise in predicting the concentration profiles of glucose, glutamine, lactate, and ammonia and protein yield over the course of the bioreactor cell culture process. Therefore, this work demonstrated the technical feasibility of real time monitoring of the bioreactor cell culture process via FTIR spectroscopy. Its implications for enabling cell culture PAT were discussed.
