大规模色谱纯化重组腺相关病毒(rAAV)工艺

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"这篇研究文章探讨了一种可扩展的色谱纯化方法，用于重组腺病毒相关病毒（rAAV）的制备，旨在解决传统CsCl梯度纯化方法在大规模生产中的不足，以及纯化后AAV载体中可能存在的细胞蛋白和腺病毒核酸污染问题。" 本文详细介绍了针对重组腺病毒相关病毒（rAAV）的一种可扩展的色谱纯化过程，这是基因治疗应用中的一种关键病毒载体。传统的AAV纯化通常依赖于CsCl梯度法，但这种方法并不适用于大规模生产，且纯化后的AAV往往含有较高的细胞蛋白和腺病毒核酸杂质。为了解决这一问题，作者们开发了一种新的、快速而高效的纯化方法，用于从原始细胞裂解液中纯化rAAV。在方法部分，文章可能会详细介绍所采用的色谱技术，如离子交换色谱、亲和色谱或凝胶过滤色谱等，并阐述每一步的具体操作，包括样品的预处理、上样、洗脱和收集纯化产物等步骤。此外，可能还会讨论优化纯化效率的关键参数，如缓冲液的组成、流速、pH值和离子强度等。实验结果部分，文章可能展示了新方法的性能，通过比较传统方法和新方法的纯化效果，如纯化产量、病毒颗粒的活性、杂质去除程度等方面的数据。这些数据可能通过电泳、紫外光谱、质谱等分析手段获得，并通过定量分析来证明新方法的优越性。讨论部分，作者可能会对实验结果进行深入分析，讨论新方法在提高纯化效率和降低杂质水平方面的优势，以及可能的优化方向。同时，他们可能还会探讨这种方法对于基因治疗产业的影响，尤其是在大规模生产rAAV疫苗或疗法时的潜在应用。结论部分，作者会总结新色谱纯化策略的优点，强调其在解决现有纯化问题上的贡献，并指出该方法在基因治疗领域工业化生产中的应用前景。此外，可能会提出未来的研究方向，如进一步提高纯化效率、降低成本，或者优化工艺以适应不同类型的rAAV。这篇学术论文提供了rAAV纯化技术的一个重要进展，对于基因治疗研究和工业生产具有重要意义，它不仅提高了纯化效率，还减少了杂质，为rAAV的临床应用铺平了道路。

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.

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