大规模重组腺相关病毒(rAAV)色谱纯化工艺的创新与应用

下载需积分: 9 | PDF格式 | 226KB | 更新于2024-08-06 | 68 浏览量 | 举报

本文是一篇关于重组腺相关病毒（rAAV）大规模色谱纯化的研究论文。rAAV是一种被开发用于基因疗法的人类小DNA病毒，因其无害且能高效地将遗传物质导入宿主细胞而受到广泛关注。传统上，rAAV的纯化方法是依赖于氯化铯梯度法，但这种方法在大规模生产中并不适用，因为它可能导致产品质量不高，且易于混杂细胞和腺病毒的蛋白质及核酸杂质。为了克服这一问题，研究者们开发了一种新的、适用于工业规模的rAAV纯化流程。该工艺专注于提高效率和纯度，旨在解决传统方法在大生产中的局限性。研究团队采用的方法可能包括亲和层析、离子交换或尺寸分离等高效色谱技术，这些技术能够有效地去除非目标产物，如细胞残留物和病毒粒子间的非特异性结合。论文作者Catherine R. O'Riordan及其同事详细描述了他们的工艺设计，可能包括优化的洗脱条件、柱子选择以及质量控制步骤，以确保纯化的rAAV具有高度的生物活性和安全性。他们还可能讨论了工艺的可扩展性和经济性，这对于任何大规模生产过程来说都是关键因素。通过接收日期、修订和接受日期以及在线发表日期，我们可以推测这是一个经过精心设计并经过多次修订后得出的成熟研究成果，旨在为rAAV的商业化生产提供一种可靠的纯化策略。这篇论文的重要性在于它不仅提供了rAAV纯化的新方法，还可能为其他基因治疗领域的工业化生产带来启示，推动了生物制药行业的技术进步。对于制药公司和研究机构而言，理解并应用这种工艺对于确保rAAV药物的质量控制和商业化成功至关重要。

Chemical, Inc., Natick, MA, USA) for 1 h at room

temperature followed by trypsin treatment 0.05% for

1 h in the presence of 1% Tween-80. The resulting lysate

was ®ltered through a 0.8 mm Millex HV ®lter unit before

chromatography. Routinely 10±15 roller bottles were

harvested equivalent to 1±5 e 9 cells.

BioRad ceramic hydroxyapatite

(80 mm)

Cell lysates from 293 cells infected with adeno-associated

virus (in the presence of Ad5ts149) were chromato-

graphed on a hydroxyapatite resin (40 ml) which was pre-

equilibrated with 10 mM sodium phosphate pH 6.4,

containing 10 mM NaCl, 0.1% Tween-80, 10% glycerol

and 2 mM MgCl

. rAAVbgal was applied to the resin in the

same buffer. Bound proteins were eluted from the resin

using a linear salt gradient (120 ml) of 10±400 mM

sodium phosphate at pH 6.4. Fractions collected from the

resin were assayed for rAAVbgal proteins by immuno-

blotting using an antibody against the three capsid

proteins of rAAVbgal, VP1, VP2 and VP3 (Catalog 03-

65158, American Research Products, Belmont, MA, USA).

Fractions eluted from the resin were also assayed for

adenoviral-contaminating proteins by immunoblotting

using an anti-adenoviral antibody.

DEAE chromatography

A DEAE Macroprep resin (BioRad) (5 ml) was equili-

brated with 10 mM sodium phosphate buffer containing

50 mM NaCl, 10% glycerol, pH 7.5. rAAVbgal-containing

fractions eluted from the hydroxyapatite column were

pooled and dialyzed into the same buffer used for

equilibration of the DEAE resin. A linear gradient

(50 mM±1 M NaCl in 10 mM sodium phosphate, pH 7.5,

10% glycerol, 0.05% Tween-80) was applied to the resin

at a ¯ow rate of 5 ml/min, the volume of the gradient was

50 ml. Bound proteins were eluted from the resin and

collected in 2.5-ml fractions. Each fraction was assayed

for rAAVbgal proteins and contaminating adenoviral

proteins (Coomassie blue staining and immunoblotting).

In addition all fractions were assayed for rAAVbgal and

Ad5ts149 infectivity by titer analysis. Fractions which

were positive for both rAAVbgal protein and infectivity

were pooled and chromatographed further using a

Cellu®ne sulphate resin.

Cellu®ne

sulphate resin (Amicon)

Cellu®ne sulphate resin (3 ml column volume) was

equilibrated with phosphate buffer saline (PBS) contain-

ing 10% glycerol. Fractions eluted from the DEAE resin

containing both rAAVbgal proteins and DNA were pooled

and applied to the resin at a ¯ow rate of 4 ml/min. The

resin was washed with 250 mM NaCl and a linear salt

gradient (0.25±1 M NaCl in PBS/10% glycerol) was

applied. The volume of the gradient was 10 ml. The

eluted fraction and the ¯ow through were analyzed for

both rAAVbgal and Ad5ts149 proteins (immunoblotting)

and infectivity (titer analysis). The ®nal fraction was also

assayed for rAAVbgal DNA by slot blot analysis.

SDS-PAGE analysis

One-dimensional SDS-PAGE was performed using

10±20% gradient (Daiichi) gels. Samples were boiled

for 5 min in SDS-PAGE reducing buffer 125 mM Tris-HCl,

pH 6.8, 20% glycerol, 4% (wt/vol) SDS, 0.05% bromo-

phenol blue, 0.5% b-mercaptoethanol before loading on

gel. Proteins in the gel were detected using Coomassie

blue or silver stain. For immunoblotting, PVDF mem-

branes (Novex) were pre-wetted with methanol and

soaked in 10 mM CAPS, pH 11.0 containing 10%

methanol. Gels were equilibrated in this transfer buffer

for 10 min and then blotted at 30 V for 1 h in a Novex Blot

Module. After transfer membranes were blocked with 1%

dried milk in TBS (20 mM Tris-HCl, pH 7.5 containing

150 mM NaCl) for 1 h. After blocking, the membranes

were probed with anti-VP1, VP2, VP3 antibody or anti-

adenoviral antibody in 20 mM Tris-HCl, 150 mM NaCl,

pH 7.5 and 0.05% Tween 20 (TBST) containing 0.1% BSA

for 1 h. The membranes were incubated with horseradish

peroxidase labeled anti-mouse or anti-goat IgG for 45 min

and the immunoreactive bands visualized by chemilumi-

nescence using the BM Chemiluminescent Western

Blotting Detection System (Boehringer Mannheim).

Measurement of infectious particles

The rAAVbgal titer was determined by an end-point

dilution assay on 293 cells. In brief, 293 cells were plated

into a 96-well micro-titer plate, 100 ml of 5 e 5 cells/ml

for each well. In a separate plate a 200-ml aliquot of virus

sample diluted 1 : 100 was added to the ®rst column and

was serially diluted two-fold across the plate. A 100 ml-

aliquot of each well was transferred to its identical

position in the 293 seeded plate and allowed to incubate

at 37uC in a humidi®ed air 5% CO

incubator for 2 days.

The media was removed by aspiration and the cells were

stained with X-gal (5 bromo-4-chloro-3-indol-b-D-

galactopyranoside) for 20±24 h. Titers were calculated

with a computer program based on Karber's method [33].

The titer of contaminating Ad5ts149 was determined

using a similar end-point dilution assay except staining

was for hexon using ¯uorescence isothiocyanate (FITC)-

conjugated anti-hexon antibody [34].

Slot blot analysis of column fractions

for detection of rAAV DNA

Column fractions were assayed for rAAVbgal DNA by slot

blot analysis as described by Vincent et al. [20].

446 C. R. O'Riordan et al.

剩余10页未读，继续阅读

weixin_38621441

粉丝: 7

大规模重组腺相关病毒(rAAV)色谱纯化工艺的创新与应用

大规模色谱纯化重组腺相关病毒(rAAV)工艺

杆状病毒表达载体系统制造 rAAV 的机制模型附matlab代码.zip

低氧启动重组腺相关病毒的构建

VEGF165低氧启动重组腺相关病毒的构建及表达

重组腺相关病毒质量控制的qPCR技术研究进展 (2014年)

重组腺相关病毒2/8的聚乙烯亚胺制备法 (2010年)

表达Kallistatin的9型重组腺相关病毒的制备及其体外表达效率 (2013年)

重组分泌型内皮抑素腺相关病毒原位治疗膀胱癌的实验研究

重构腺相关病毒转导PDX-1对糖尿病模型鼠的影响

rAAV-hGAD65重组载体的构建和体外功能检测

最新资源