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The European Union Biological Standardization Program: Detailed Characterization of GMP-produced Recombinant Bet v 1.0101 and Phl p 5.0109 as Candidate Reference Standards
S216 Abstracts
831
MONDAY
The European Union Biological Standardization Program: Detailed Characterization of GMP-produced Recombinant Bet v 1.0101 and Phl p 5.0109 as Candidate Reference Standards M. Himly1, A. Neubauer2, P. Moingeon3, O. Cromwell4, R. van Ree5, K. Buchheit6, S. Vieths7, F. Ferreira1; 1Christian Doppler Laboratory for Allergy Diagnosis and Therapy, University of Salzburg, Salzburg, Austria, 2 Biomay AG, Vienna, Austria, 3Research and Development, Stallergenes SA, Antony, France, 4Allergopharma, Joachim Ganzer KG, Reinbek, Germany, 5Academic Medical Center, Amsterdam, Netherlands, 6European Directorate for Quality of Medicines and Healthcare, Strasbourg, France, 7 Dept of Allergology, Paul-Ehrlich-Institut, Langen, Germany. RATIONALE: Standardization of allergen extracts requires availability of well-characterized recombinant allergens used as reference standards provided by the European regulatory authorities. The objective of this study was the detailed physicochemical and immunological characterization of rBet v 1.0101 and rPhl p 5.0109, which shall be used in a ring trial within the framework of the Biological Standardization Program BSP090 of the European Directorate for Quality of Medicines and Healthcare (EDQM). METHODS: Recombinant Bet v 1.0101 and Phl p 5.0109 were produced under GMP conditions and analyzed by an array of physicochemical and immunological methods for identity, quantity, homogeneity, folding and denaturation, aggregation state and stability in solution, alum-adsorbed suspension, and lyophilized state, as well as for biological activity. RESULTS: Using mass spectrometry both preparations were shown to contain rBet v 1.0101 and rPhl p 5.0109, respectively. SDS-PAGE, isoelectric focussing, deamidation analysis, and size-exclusion chromatography with light scattering revealed sample homogeneity of >99.9%. Upon storage at 148 C they retained monomeric state up to three months. Proteins were quantified by amino acid analysis. Folding studies based on Fourier Transform-infrared spectroscopy revealed similar secondary structure and aggregation behavior in solution, alum-adsorbed suspension, and lyophilized state. Biological activity of both proteins was comparable to their natural counterparts. CONCLUSIONS: Recombinant Bet v 1.0101 and Phl p 5.0109 were characterized extensively by physicochemical and immunological methods. They were shown highly stable, monomeric, and immunologically equivalent to their natural counterparts. Thus, they represent appropriate candidate reference standards for allergen products of birch and timothy grass.
832
Bacterial DNA Sequences Isolated from Standardized Dust Mite Extracts and Wild Mites C. Valerio1, L. G. Arlian2, J. E. Slater1; 1FDA/CBER/OVRR/DBPAP, Rockville, MD, 2Wright State University, Dayton, OH. RATIONALE: D. farinae extracts contain more endotoxin than D. pteronyssinus extracts. We have shown that this endotoxin is associated with bacterial DNA in D. farinae mites. Sequence analysis suggests that this bacterial DNA is from Bartonella. In this study, we have probed for bacterial DNA in D. farinae extracts and other mite species. METHODS: We tested 27 lots of mite extracts (13 lots D. farinae and 14 lots D. pteronyssinus) obtained from six US manufacturers, as well as 10 non-mite extracts (cat hair/pelt, cockroach, honeybee, Bermuda and ryegrass). In addition, we tested whole mite preparations from five additional species (C. arcuatus, L. destructor, E. maynei, A. siro, and T. putrescentiae,). DNA from commercial extracts (extracted with QIAamp DNA Minikit) and DNA from whole mites (extracted with DNAzol), was amplified as previously described using high fidelity PCR Supermix (Invitrogen). Sequencing was performed on PCR product from D. farinae extracts (5 different lots) and 44 whole mite DNA preparations. The sequences were analyzed for homologies by BLAST. RESULTS: Among the mite extracts, bacterial DNA was identified in 6/13 D. farinae extracts but 0/14 D. pteronyssinus extracts. Among the non-mite extracts, bacterial DNA was identified in only 1/10 (German cockroach). Among the non-Dermatophagoides mite preparations, bacterial DNA was detected in all five. However, Bartonella homologies were detected only in C. arcuatus.
J ALLERGY CLIN IMMUNOL FEBRUARY 2009
CONCLUSIONS: Consistent with our previous findings with whole mites, bacterial DNA may be detected in D. farinae but not D. pteronyssinus allergen extracts. In addition, bacterial DNA of different homologies are detected in other mite species.
833
Significant Reduction In Combined Symptom And Medication Score Compared With Placebo Following MPL-Adjuvanted uSCIT In Patients With Seasonal Grass Pollen Allergy L. M. DuBuske1, M. Castells1, T. Holdich2; 1Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, 2Allergy Therapeutics, Worthing, United Kingdom. RATIONALE: The efficacy and tolerability of ultra short course subcutaneous immunotherapy (uSCIT) adjuvanted with monophosphoryl lipid A (MPL), a Toll-like Receptor (TLR) 4 agonist, in the treatment of seasonal allergic rhinoconjunctivitis (SAR) to grass pollen was assessed. METHODS: This multicenter, double-blind, placebo-controlled study included 1028 grass pollen-allergic subjects. Subjects received a pre-seasonal, 4-injection course of uSCIT containing a 13 grass pollen allergoid (300, 800, 2000, and 2000 SU) plus 50 mg MPL per injection in an L-tyrosine depot (Pollinex QuattroÒ) at approximately weekly intervals. Efficacy was assessed by combined symptom and medication score (CSMS) using patient diaries completed throughout the grass pollen season. Local and systemic reactions were recorded during the 24 hours following each injection. RESULTS: There was a 13.4% improvement (p 5 0.0038) in CSMS in patients who received uSCIT (n 5 514) compared with placebo (n 5 513) over the four peak pollen weeks. In those subjects who had no missing data during the peak pollen period, the improvement in CSMS was 26.9% for uSCIT compared with placebo (p 5 0.0031). Adverse events (AEs) were predominantly local and discontinuations due to AEs were infrequent (1.4% for uSCIT, 0.8% for placebo), with no treatment-related anaphylaxis. Following each injection, >60% of treated patients experienced no AEs. CONCLUSIONS: Pre-seasonal uSCIT was highly effective and well tolerated, with a clinically significant reduction in SAR symptoms. Adverse events were predominantly local and mild to moderate in nature, indicating a favourable tolerability profile. This is the first large Phase III study of specific allergen immunotherapy for SAR to meet its primary efficacy endpoint.
834
Interactive Effect of Eczema and Food Allergy on Asthma in Children D. M. Caruso, L. M. Arguelles, J. S. Kim, R. Gupta, A. Schroeder, K. E. Meyer, Y. Vucic, B. Rowland, E. Frankis, J. Costello, A. Shelton, J. A. Pongracic, X. Wang; Children’s Memorial Hospital, Chicago, IL. RATIONALE: The atopic march is well documented, but the inter-relationship of eczema, food allergy (FA), and asthma is not well understood. METHODS: This analysis included 512 children > 5 6 yrs from a familybased food allergy cohort in Chicago. Eczema, FA and asthma were based on parental report of physician-diagnosis by questionnaire interview. FA was further verified by timing of reaction, clinical symptoms, and corroborative sIgE and skin prick test. Logistic regression nd survival analysis estimated the risk and early onset of asthma in relation to eczema, adjusting for age, gender, household income, months in childcare, presence of FA, maternal education, parental atopy, and intrafamilial correlation. The interactive effect of eczema and FA on asthma was also examined. RESULTS: The prevalence of eczema was 44.7%, FA 32.0%, and asthma 39.5, Eczema was strongly associated with asthma but the association was significantly modified by FA. Compared to those with neither eczema nor FA, those with eczema but no FA had higher risk of asthma (OR 5 3.1, 95%CI 1.8, 5.3). The highest risk of asthma was found in those with both eczema and FA (OR 5 9.0, 95%CI: 5.1, 15.9). Test of interaction between eczema and FA on asthma was significant (P 5 0.0005). The median age of onset for eczema and FAwas 1 year and for asthma, 3 years. Children with eczema developed asthma earlier than children without eczema (Hazard Ratio 5 1.6, 95%CI 1.1, 2.5). CONCLUSIONS: Eczema and FA interactively increased the risk of asthma in this sample. Additional longitudinal studies are needed to elucidate the biological mechanisms underlying this association.
831
MONDAY
The European Union Biological Standardization Program: Detailed Characterization of GMP-produced Recombinant Bet v 1.0101 and Phl p 5.0109 as Candidate Reference Standards M. Himly1, A. Neubauer2, P. Moingeon3, O. Cromwell4, R. van Ree5, K. Buchheit6, S. Vieths7, F. Ferreira1; 1Christian Doppler Laboratory for Allergy Diagnosis and Therapy, University of Salzburg, Salzburg, Austria, 2 Biomay AG, Vienna, Austria, 3Research and Development, Stallergenes SA, Antony, France, 4Allergopharma, Joachim Ganzer KG, Reinbek, Germany, 5Academic Medical Center, Amsterdam, Netherlands, 6European Directorate for Quality of Medicines and Healthcare, Strasbourg, France, 7 Dept of Allergology, Paul-Ehrlich-Institut, Langen, Germany. RATIONALE: Standardization of allergen extracts requires availability of well-characterized recombinant allergens used as reference standards provided by the European regulatory authorities. The objective of this study was the detailed physicochemical and immunological characterization of rBet v 1.0101 and rPhl p 5.0109, which shall be used in a ring trial within the framework of the Biological Standardization Program BSP090 of the European Directorate for Quality of Medicines and Healthcare (EDQM). METHODS: Recombinant Bet v 1.0101 and Phl p 5.0109 were produced under GMP conditions and analyzed by an array of physicochemical and immunological methods for identity, quantity, homogeneity, folding and denaturation, aggregation state and stability in solution, alum-adsorbed suspension, and lyophilized state, as well as for biological activity. RESULTS: Using mass spectrometry both preparations were shown to contain rBet v 1.0101 and rPhl p 5.0109, respectively. SDS-PAGE, isoelectric focussing, deamidation analysis, and size-exclusion chromatography with light scattering revealed sample homogeneity of >99.9%. Upon storage at 148 C they retained monomeric state up to three months. Proteins were quantified by amino acid analysis. Folding studies based on Fourier Transform-infrared spectroscopy revealed similar secondary structure and aggregation behavior in solution, alum-adsorbed suspension, and lyophilized state. Biological activity of both proteins was comparable to their natural counterparts. CONCLUSIONS: Recombinant Bet v 1.0101 and Phl p 5.0109 were characterized extensively by physicochemical and immunological methods. They were shown highly stable, monomeric, and immunologically equivalent to their natural counterparts. Thus, they represent appropriate candidate reference standards for allergen products of birch and timothy grass.
832
Bacterial DNA Sequences Isolated from Standardized Dust Mite Extracts and Wild Mites C. Valerio1, L. G. Arlian2, J. E. Slater1; 1FDA/CBER/OVRR/DBPAP, Rockville, MD, 2Wright State University, Dayton, OH. RATIONALE: D. farinae extracts contain more endotoxin than D. pteronyssinus extracts. We have shown that this endotoxin is associated with bacterial DNA in D. farinae mites. Sequence analysis suggests that this bacterial DNA is from Bartonella. In this study, we have probed for bacterial DNA in D. farinae extracts and other mite species. METHODS: We tested 27 lots of mite extracts (13 lots D. farinae and 14 lots D. pteronyssinus) obtained from six US manufacturers, as well as 10 non-mite extracts (cat hair/pelt, cockroach, honeybee, Bermuda and ryegrass). In addition, we tested whole mite preparations from five additional species (C. arcuatus, L. destructor, E. maynei, A. siro, and T. putrescentiae,). DNA from commercial extracts (extracted with QIAamp DNA Minikit) and DNA from whole mites (extracted with DNAzol), was amplified as previously described using high fidelity PCR Supermix (Invitrogen). Sequencing was performed on PCR product from D. farinae extracts (5 different lots) and 44 whole mite DNA preparations. The sequences were analyzed for homologies by BLAST. RESULTS: Among the mite extracts, bacterial DNA was identified in 6/13 D. farinae extracts but 0/14 D. pteronyssinus extracts. Among the non-mite extracts, bacterial DNA was identified in only 1/10 (German cockroach). Among the non-Dermatophagoides mite preparations, bacterial DNA was detected in all five. However, Bartonella homologies were detected only in C. arcuatus.
J ALLERGY CLIN IMMUNOL FEBRUARY 2009
CONCLUSIONS: Consistent with our previous findings with whole mites, bacterial DNA may be detected in D. farinae but not D. pteronyssinus allergen extracts. In addition, bacterial DNA of different homologies are detected in other mite species.
833
Significant Reduction In Combined Symptom And Medication Score Compared With Placebo Following MPL-Adjuvanted uSCIT In Patients With Seasonal Grass Pollen Allergy L. M. DuBuske1, M. Castells1, T. Holdich2; 1Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, 2Allergy Therapeutics, Worthing, United Kingdom. RATIONALE: The efficacy and tolerability of ultra short course subcutaneous immunotherapy (uSCIT) adjuvanted with monophosphoryl lipid A (MPL), a Toll-like Receptor (TLR) 4 agonist, in the treatment of seasonal allergic rhinoconjunctivitis (SAR) to grass pollen was assessed. METHODS: This multicenter, double-blind, placebo-controlled study included 1028 grass pollen-allergic subjects. Subjects received a pre-seasonal, 4-injection course of uSCIT containing a 13 grass pollen allergoid (300, 800, 2000, and 2000 SU) plus 50 mg MPL per injection in an L-tyrosine depot (Pollinex QuattroÒ) at approximately weekly intervals. Efficacy was assessed by combined symptom and medication score (CSMS) using patient diaries completed throughout the grass pollen season. Local and systemic reactions were recorded during the 24 hours following each injection. RESULTS: There was a 13.4% improvement (p 5 0.0038) in CSMS in patients who received uSCIT (n 5 514) compared with placebo (n 5 513) over the four peak pollen weeks. In those subjects who had no missing data during the peak pollen period, the improvement in CSMS was 26.9% for uSCIT compared with placebo (p 5 0.0031). Adverse events (AEs) were predominantly local and discontinuations due to AEs were infrequent (1.4% for uSCIT, 0.8% for placebo), with no treatment-related anaphylaxis. Following each injection, >60% of treated patients experienced no AEs. CONCLUSIONS: Pre-seasonal uSCIT was highly effective and well tolerated, with a clinically significant reduction in SAR symptoms. Adverse events were predominantly local and mild to moderate in nature, indicating a favourable tolerability profile. This is the first large Phase III study of specific allergen immunotherapy for SAR to meet its primary efficacy endpoint.
834
Interactive Effect of Eczema and Food Allergy on Asthma in Children D. M. Caruso, L. M. Arguelles, J. S. Kim, R. Gupta, A. Schroeder, K. E. Meyer, Y. Vucic, B. Rowland, E. Frankis, J. Costello, A. Shelton, J. A. Pongracic, X. Wang; Children’s Memorial Hospital, Chicago, IL. RATIONALE: The atopic march is well documented, but the inter-relationship of eczema, food allergy (FA), and asthma is not well understood. METHODS: This analysis included 512 children > 5 6 yrs from a familybased food allergy cohort in Chicago. Eczema, FA and asthma were based on parental report of physician-diagnosis by questionnaire interview. FA was further verified by timing of reaction, clinical symptoms, and corroborative sIgE and skin prick test. Logistic regression nd survival analysis estimated the risk and early onset of asthma in relation to eczema, adjusting for age, gender, household income, months in childcare, presence of FA, maternal education, parental atopy, and intrafamilial correlation. The interactive effect of eczema and FA on asthma was also examined. RESULTS: The prevalence of eczema was 44.7%, FA 32.0%, and asthma 39.5, Eczema was strongly associated with asthma but the association was significantly modified by FA. Compared to those with neither eczema nor FA, those with eczema but no FA had higher risk of asthma (OR 5 3.1, 95%CI 1.8, 5.3). The highest risk of asthma was found in those with both eczema and FA (OR 5 9.0, 95%CI: 5.1, 15.9). Test of interaction between eczema and FA on asthma was significant (P 5 0.0005). The median age of onset for eczema and FAwas 1 year and for asthma, 3 years. Children with eczema developed asthma earlier than children without eczema (Hazard Ratio 5 1.6, 95%CI 1.1, 2.5). CONCLUSIONS: Eczema and FA interactively increased the risk of asthma in this sample. Additional longitudinal studies are needed to elucidate the biological mechanisms underlying this association.