| Co-Investigator(Kenkyū-buntansha) |
OHKUMA Kunio The Chemo-Sero-Therapeutic Research Institute, General Manager, 第一製造部, 上席研究員
SAKATA Masayo Kumamoto University, Applied Chemistry, Assistant Professor, 工学部, 助手 (60187391)
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| Budget Amount *help |
¥8,500,000 (Direct Cost: ¥8,500,000)
Fiscal Year 2002: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 2001: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2000: ¥4,400,000 (Direct Cost: ¥4,400,000)
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| Research Abstract |
To remove nucleic acids from a solution of a cell product used as a drug, animated poly(γ-metbyl L-glutamate) (PMLG-NH_2) particles and cross-linked poly(ethyleneimine) (PH) spherical particles were prepared. The particles, which had diameters of 44 to 105 μm were used as adsorbents. The adsorption of DNA and cell products to the adsorbents were determined using a batchwise method. The adsorbing activity for nucleic acid (purified DNA from salmon spermary) increased with increase in either the amino-group content or pore-size (molecular mass exclusion of polysaccharide, M_<lim>) of the adsorbent.
The apparent dissociation constant (K_d) between the DNA and the PMLG-NH_2 adsorbent (amino-group content: 3.5 meq/g) decreased from 8.2 × 10^<-13> mol/l (M) to 2.0 × 10^<-13> M with increase in pore size from 2 × 10^3 to 2 × 10^5, at pH 7.0 and an ionic strength of μ = 0.05. On the other hand, the adsorbing activity of bovine serum albumin (BSA) increased with increasing M_<lim> of the particl
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es, but decreased with increasing ionic strength of the buffer. As a result, when M_<lim> was 2 × 10^3 and amino-group content was 3.5 meq/ml, the PMLG-NH_2 removed DNA from various DNA-containing samples, such as protein solutions, at pH 7.0 and ionic strength (μ) of 0.2.
The K_d between the DNA and the PEI adsorbent decreased from 8.5 × 10^<-8> to 9.5 × 10^<-10> M with an increase in PEI ratio from 50 to 90 unit-mol% under physiological conditions (ionic strength of μ = 0.2, pH 7.0). On the other hand, the adsorbing activity of BSA also increased with increasing PEI ratio of the adsorbent from 70 unit-mol% or higher, but sharply decreased with increasing buffer's ionic strength. The adsorbing activity of γ-globulin increased with decreasing PEI ratio to 70 unit-mol% or lower. As a result, when the PEI adsorbent, having a PEI ratio of 80 unit-mol%, were used as the adsorbent, they only selectively removed DNA from various protein solutions at an ionic strength of μ = 0.2 and a pH of 7.0. The adsorbent decreased the concentration of DNA in each protein solution to less than 10 ng/ ml, and the recovery rate of protein was more 97% in all cases. Less
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