title: Evaluation of dry blood spot technique for quantification of an Anti-CD20 monoclonal antibody drug in human blood samples.
abstract: BACKGROUND
  To evaluate the dried blood spot (DBS) technique in ELISA quantification of larger biomolecular drugs, an anti-CD20 monoclonal antibody drug was used as an example. A method for the quantification of the anti-CD20 drug in human DBS was developed and validated.
  
  
  METHODS
  The drug standard and quality control samples prepared in fresh human blood were spotted on DBS cards and then extracted. A luminescent ELISA was used for quantification of the drug from DBS samples.
  
  
  RESULTS
  The assay range of the anti-CD20 drug standards in DBS was 100-2500ng/mL. The intra-assay precision (%CV) ranged from 0.4% to 10.1%, and the accuracy (%Recovery) ranged from 77.9% to 113.9%. The inter assay precision (%CV) ranged from 5.9% to 17.4%, and the accuracy ranged from 81.5% to 110.5%. The DBS samples diluted 500 and 50-fold yielded recovery of 88.7% and 90.7%, respectively. The preparation of DBS in higher and lower hematocrit (53% and 35%) conditions did not affect the recovery of the drug. Furthermore, the storage stability of the anti-CD20 drug on DBS cards was tested at various conditions. It was found that the anti-CD20 drug was stable for one week in DBS stored at room temperature. However, it was determined that the stability was compro]mised in DBS stored at high humidity, high temperature (55°C), and exposed to direct daylight for a week, as well as for samples stored at room temperature and high humidity conditions for a month. Stability did not change significantly in samples that underwent 3 freeze/thaw cycles.
  
  
  CONCLUSIONS
  Our results demonstrated a successful use of DBS technique in ELISA quantification of an anti-CD20 monoclonal antibody drug in human blood. The stability data provides information regarding sample storage and shipping for future clinical studies. It is, therefore, concluded that the DBS technique is applicable in the quantification of other large biomolecule drugs or biomarkers.
authors.affiliation: Alliance Pharma, Inc., 17 Lee Blvd. Malvern, PA 19355, USA.
authors.forename: Yong-Qing
authors.initials: YQ
authors.lastname: Lin
authors.forename: Yilu
authors.initials: Y
authors.lastname: Zhang
authors.forename: Connie
authors.initials: C
authors.lastname: Li
authors.forename: Louis
authors.initials: L
authors.lastname: Li
authors.forename: Kelley
authors.initials: K
authors.lastname: Zhang
authors.forename: Shawn
authors.initials: S
authors.lastname: Li
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000276:immunology; D018951:Antigens, CD20 / Q000276:immunology*; D059788:Dried Blood Spot Testing / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005615:Freezing; D006801:Humans; D006813:Humidity; D051379:Mice; D015203:Reproducibility of Results; D013048:Specimen Handling; D013696:Temperature; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D018951:Antigens, CD20