tetano
Editor, Senior Moderator
Vaccine. 2014 Mar 10. pii: S0264-410X(14)00205-9. doi: 10.1016/j.vaccine.2014.02.032. [Epub ahead of print]
Sensitive methods for evaluation of antibodies for host cell protein analysis and screening of impurities in a vaccine process.
Lundstr?m CS1, Mattsson A1, L?vgren K2, Eriksson A1, Sundberg AL1, Lundgren M1, Nilsson CE3.
Author information
Abstract
BACKGROUND:
Host cell proteins (HCP) should be carefully monitored in vaccine production. To achieve a reliable HCP estimation, a mixture of polyclonal antibodies (pAbs) with broad affinity would be of preference. Sensitive evaluations of the pAbs are therefore of value.
METHODS:
Column purification of specific HCPs with affinity to the anti-HCP pAbs was compared with Western blotting of the anti-HCP pAbs binding to filter bound total lysate. The anti-HCP pAbs were used in an HCP quantification analysis using surface plasmon resonance (SPR). Host cell derived impurities from an influenza vaccine process were analyzed using 2-D DIGE analysis.
RESULTS:
The Western blotting showed a similar HCP binding pattern of anti-HCP pAbs from immunizations using two adjuvants: CFA/IFA and AbISCO?. From the column purification of HCPs, total proteins detectable were similar for all anti-HCP pAbs; however the immune response pattern differed significantly for the anti-HCP pAbs from the AbISCO? immunization. In the SPR HCP quantification assay the standard curve ranged from 0.3 to 40μg/ml. The advantage of SPR compared with ELISA was the decreased hands on time and that the sample number was not limiting. The 2-D DIGE showed that most of the HCPs were removed at the clarification and virus capture step.
DISCUSSION:
Column purification of HCPs with affinity to the anti-HCP pAbs increased the sensitivity of affinity analysis compared with Western blotting and opened the possibility of further analysis. The anti-HCP pAbs did not interact with proteins in the virus; simplifying analysis of process samples using SPR. 2-D DIGE analysis gave a direct study of the impurity profile with the advantage of independence from antibody performance.
Copyright ? 2014 Elsevier Ltd. All rights reserved.
KEYWORDS:
2-D DIGE, Adjuvant, Biacore, Host cell protein, Influenza virus, SPR
PMID:
24631079
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/24631079
Sensitive methods for evaluation of antibodies for host cell protein analysis and screening of impurities in a vaccine process.
Lundstr?m CS1, Mattsson A1, L?vgren K2, Eriksson A1, Sundberg AL1, Lundgren M1, Nilsson CE3.
Author information
Abstract
BACKGROUND:
Host cell proteins (HCP) should be carefully monitored in vaccine production. To achieve a reliable HCP estimation, a mixture of polyclonal antibodies (pAbs) with broad affinity would be of preference. Sensitive evaluations of the pAbs are therefore of value.
METHODS:
Column purification of specific HCPs with affinity to the anti-HCP pAbs was compared with Western blotting of the anti-HCP pAbs binding to filter bound total lysate. The anti-HCP pAbs were used in an HCP quantification analysis using surface plasmon resonance (SPR). Host cell derived impurities from an influenza vaccine process were analyzed using 2-D DIGE analysis.
RESULTS:
The Western blotting showed a similar HCP binding pattern of anti-HCP pAbs from immunizations using two adjuvants: CFA/IFA and AbISCO?. From the column purification of HCPs, total proteins detectable were similar for all anti-HCP pAbs; however the immune response pattern differed significantly for the anti-HCP pAbs from the AbISCO? immunization. In the SPR HCP quantification assay the standard curve ranged from 0.3 to 40μg/ml. The advantage of SPR compared with ELISA was the decreased hands on time and that the sample number was not limiting. The 2-D DIGE showed that most of the HCPs were removed at the clarification and virus capture step.
DISCUSSION:
Column purification of HCPs with affinity to the anti-HCP pAbs increased the sensitivity of affinity analysis compared with Western blotting and opened the possibility of further analysis. The anti-HCP pAbs did not interact with proteins in the virus; simplifying analysis of process samples using SPR. 2-D DIGE analysis gave a direct study of the impurity profile with the advantage of independence from antibody performance.
Copyright ? 2014 Elsevier Ltd. All rights reserved.
KEYWORDS:
2-D DIGE, Adjuvant, Biacore, Host cell protein, Influenza virus, SPR
PMID:
24631079
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/24631079