Column-based Purification Research Articles

High purity and recovery of native filamentous hemagglutinin (FHA) from Bordetella pertussis using affinity chromatography

Filamentous hemagglutinin (FHA) is a critical adhesion molecule produced by Bordetella pertussis (BP), the causative agent of highly contagious respiratory infection known as whooping cough. FHA plays a pivotal role in the pathogenesis of whooping cough and is a key component of acellular pertussis vaccines (aPV). However, conventional purification methods for FHA often involve labor-intensive processes and result in low purity and recovery rates. Therefore, this study explores the use of monoclonal and polyclonal antibodies as specific tools to achieve highly pure and efficient FHA purification. To generate FHA-specific antibodies, polyclonal antibodies were produced by immunizing sheep and monoclonal antibodies (MAbs) were generated by immunizing mice with recombinant and native FHA. The MAbs were selected based on affinity, isotypes, and specificity, which were assessed through ELISA and Western blot assays. Two immunoaffinity columns, one monoclonal and one polyclonal, were prepared for FHA antigen purification. The purity and recovery rates of these purifications were determined using ELISA, SDS-PAGE, and immunoblotting. Furthermore, the MAbs were employed to develop an ELISA assay for FHA antigen concentration determination. The study's findings revealed that immunoaffinity column-based purification of FHA resulted in a highly pure antigen with recovery rates of approximately 57% ± 6.5% and 59% ± 7.9% for monoclonal and polyclonal columns, respectively. Additionally, the developed ELISA exhibited appropriate reactivity for determining FHA antigen concentration. This research demonstrates that affinity chromatography is a viable and advantageous method for purifying FHA, offering superior purity and recovery rates compared to traditional techniques. This approach provides a practical alternative for FHA purification in the context of aPV development.

A semi-automated and high-throughput approach for the detection of honey bee viruses in bee samples.

Deformed wing virus (DWV) was first detected in dead honey bees in 1982 but has been in honey bees for at least 300 years. Due to its high prevalence and virulence, they have been linked with the ongoing decline in honey bee populations worldwide. A rapid, simple, semi-automated, high-throughput, and cost-effective method of screening colonies for viruses would benefit bee research and the beekeeping industry. Here we describe a semi-automated approach that combines an RNA-grade liquid homogenizer followed by magnetic bead capture for total virus nucleic acid extraction. We compare it to the more commonly applied nucleic acid column-based purification method and use qPCR plus Oxford Nanopore Technologies sequencing to evaluate the accuracy of analytical results for both methods. Our results showed high reproducibility and accuracy for both approaches. The semi-automated method described here allows for faster screening of viral loads in units of 96 samples at a time. We developed this method to monitor viral loads in honey bee colonies, but it could be easily applied for any PCR or genomic-based screening assays.

Evaluation of low-cost SARS-CoV-2 RNA purification methods for viral quantification by RT-qPCR and next-generation sequencing analysis: Implications for wider wastewater-based epidemiology adoption

WastewaterBased Epidemiology (WBE) consists of quantifying biomarkers in sewerage systems to derive real-time information on the health and/or lifestyle of the contributing population. WBE usefulness was vastly demonstrated in the context of the COVID-19 pandemic. Many methods for SARS-CoV-2 RNA determination in wastewater were devised, which vary in cost, infrastructure requirements and sensitivity. For most developing countries, implementing WBE for viral outbreaks, such as that of SARS-CoV-2, proved challenging due to budget, reagent availability and infrastructure constraints. In this study, we assessed low-cost methods for SARS-CoV-2 RNA quantification by RT-qPCR, and performed variant identification by NGS in wastewater samples. Results showed that the effect of adjusting pH to 4 and/or adding MgCl2 (25 mM) was negligible when using the adsorption-elution method, as well as basal physicochemical parameters in the sample. In addition, results supported the standardized use of linear rather than plasmid DNA for a more accurate viral RT-qPCR estimation. The modified TRIzol-based purification method in this study yielded comparable RT-qPCR estimation to a column-based approach, but provided better NGS results, suggesting that column-based purification for viral analysis should be revised. Overall, this work provides evaluation of a robust, sensitive and cost-effective method for SARS-CoV-2 RNA analysis that could be implemented for other viruses, for a wider WEB adoption.

A Simplified Method for RNA Isolation from Biofabricating Hydroxyapatite Scaffolds and Identification of Appropriate Reference Genes

PurposeTo validate a simplified RNA isolation method from biofabricating hydroxyapatite (HAp) scaffolds seeded with mesenchymal stem cells (MSCs) and to identify the appropriate reference gene.MethodsTen MSCs-HAp composites were used for RNA isolation by methods based on simplified homogenization steps and column-based purification procedures, while the remaining RNA (n = 13) was extracted by traditional single-step isolation methods. The differences between the two procedures regarding the operation time, RNA quantity and quality were evaluated. Quantitative real-time PCR (qRT-PCR) analysis was performed to identify the appropriate reference gene.ResultsThe simplified method showed significant superiority in operation time (P < 0.001), RNA concentration (P < 0.001), A260/280 ratio (P = 0.005) and A260/230 ratio (P < 0.001). The average integrity number and 28 s/18 s ratio of RNA yielded by the simplified method were 9.1 ± 0.2 and 1.3 ± 0.1, respectively. The qRT-PCR analysis results indicated that the cycle threshold (Ct) values of GAPDH were significantly higher than those of the remaining 2 reference genes (ACTB and RPL13A) in the RNA samples obtained by the simplified and traditional methods (P < 0.05). The standard deviations of the ΔCt value (the difference between the Ct value and the minimum) of ACTB were higher than those of GAPDH or RPL13A, regardless of the RNA isolation method.ConclusionThe simplified method could extract intact RNA from biofabricating MSCs-HAp scaffolds and was superior to the traditional single-step procedure in operation time, RNA quantity and quality. GAPDH was identified as the most appropriate reference gene in MSCs-HAp scaffold composites due to its high quantity and good stability.

A new device-mediated miniprep method

Small-scale plasmid DNA preparation or miniprep is a fundamental technique in estimation cloning experiments and is widely used for DNA methylation analysis in epigenetic research. Current plasmid DNA minipreps use the alkali-SDS-based method in a three-solution format and require spin column-based purification steps. This procedure requires the vortexing or pipetting of pelleted bacteria by centrifugation and manual mixing of the solutions. Here, we describe a centrifuge/mixer-based instrument with the ability to perform centrifugation, vibration, and rotor oscillation in order to perform all steps of plasmid DNA isolation by device only. We found that by applying rotor oscillation-driven mixing of solutions added in the lysis and neutralization steps, homogeneous mixing was achieved within 5 s at a rotor oscillation amplitude of 45° and oscillation frequency of 400 ± 30 rpm, yielding the maximal quantity and quality of plasmid DNA. No increase in host chromosome presence purified by this approach occurs for high-copy-number plasmids compared to manually performed miniprep, and indeed, there is a significant decrease in the presence of the chromosomal fraction in low-copy-number plasmids. The supercoiled form of plasmid DNA purified at a rotor oscillation amplitude of 45° does not turn into an open circular (OC) isoform when the plasmid is stored for 1 year at plus four degrees, in contrast to the plasmid purified with rotor oscillation amplitudes of 270°, 180° and 90°. The programmed time-work-efficient protocol of plasmid miniprep installed in the device gives the extreme simplicity of plasmid minipreps speeding up and facilitating the isolation of plasmid DNAs.

Direct Detection of Extracellular Vesicle miRNAs Using a Single-Step RT-qPCR Assay.

Extracellular vesicles (EVs) are biological carriers, and EV-associated miRNAs (EV-miRNAs) are considered as a novel biomarker in multiple diseases. Currently, the column-based purification method is used to purify miRNAs from EVs. However, this method of purification is complex, time-consuming, and expensive. Therefore, a simple and cost-effective single-step quantitative reverse transcription-polymerase chain reaction (RT-qPCR) method is required to detect the expression of EV-miRNAs. This chapter describes a protocol for directly analyzing the EV-miRNAs expression from mouse bronchoalveolar lavage fluid (BALF) and serum without going for an RNA isolation and purification step from EVs. It is an efficient method in several terms such as cost-wise, time, low expertise, and accuracy in results. This method may be helpful in diagnostic blood tests used in medical centers or research laboratories.

Purification of monoclonal antibody using cation exchange z2 laterally-fed membrane chromatography – A potential alternative to protein A affinity chromatography

Protein A affinity chromatography, which is the standard method for purifying monoclonal antibodies (mAbs) is expensive and involves elution at acidic pH, which could degrade the mAb. Moreover, it cannot be used to fractionate charge variants or to remove mAb aggregates. In this study, we examine the purification of Trastuzumab by cation-exchange z2 laterally-fed membrane chromatography (or z2LFMC). It has been shown that z2LFMC is suitable for carrying out high-speed, high-resolution protein purification. The results discussed in this paper demonstrate that the purity of Trastuzumab obtained by the z2LFMC process was comparable to that obtained by protein A chromatography (i.e., >95% with both methods), while the recovery was significantly greater with z2LFMC (90.4% as opposed to 81.7%). Also, the z2LFMC process was faster, did not require acidic pH conditions for elution, and the mAb was eluted in 2.24 bed volumes as opposed to 2.74 bed volumes with protein A chromatography. The mAb productivity obtained with the z2LFMC process was more than 3 times higher than that with the column-based purification process. The z2LFMC device is significantly cheaper than its equivalent protein A affinity column, and earlier studies have shown it to be suitable for separating mAb charge variants and aggregates. Therefore, the z2LFMC process could be evaluated as a cheaper and more efficient alternative to protein A affinity chromatography.

DIE-RNA: A Reproducible Strategy for the Digestion of Normal and Injured Pancreas, Isolation of Pancreatic Cells from Genetically Engineered Mouse Models and Extraction of High Quality RNA.

The isolation of ribonucleic acid (RNA) suitable for gene expression studies is challenging in the pancreas, due to its high ribonuclease activity. This is even more complicated during pancreatitis, a condition associated with inflammation and fibrosis. Our aim was to implement a time-effective and reproducible protocol to isolate high quality RNA from specific pancreatic cell subtypes, in normal and inflammatory conditions. We used two genetically engineered mouse models (GEMM), Ela-CreER/YFP and Sox9-CreER/YFP, to isolate acinar and ductal cells, respectively. To induce pancreatitis, mice received a caerulein treatment (125 μg/kg) for 8 and 72 h. We alternatively used EGTA and calcium buffers that contain collagenase P (0.6 mg/mL) to rapidly digest the pancreas into individual cells. Most of the cells from normal and injured pancreas were single-dissociated, exhibited a round morphology and did not incorporate trypan blue dye. Cell suspensions from Ela- and Sox9-CreER/YFP pancreas were then sorted by flow cytometry to isolate the YFP-positive acinar and ductal cells, respectively. Sorted cells kept a round shape and emitted fluorescence detected by the 38 HE green fluorescence filter. RNA was isolated by column-based purification approach. The RNA integrity number (RIN) was high in sorted acinar cell fractions treated with or without caerulein (8.6 ± 0.17 and 8.4 ± 0.09, respectively), compared to the whole pancreas fraction (4.8 ± 1.1). Given the low number of sorted ductal cells, the RIN value was slightly lower compared to acini (7.4 ± 0.4). Quantitative-PCR experiments indicated that sorted acinar and ductal cells express the specific acinar and ductal markers, respectively. Additionally, RNA preparations from caerulein-treated acinar cells were free from significant contamination with immune cell RNA. We thus validated the DIE (Digestion, Isolation, and Extraction)-RNA tool as a reproducible and efficient protocol to isolate pure acinar and ductal cells in vivo and to extract high quality RNA from these cells.

One-step integrated clarification and purification of a monoclonal antibody using Protein A Mag Sepharose beads and a cGMP-compliant high-gradient magnetic separator

Monoclonal antibodies are a dominant component of today's biopharmaceutical market and are typically purified by classical platform processes. However, high costs and rising demands are drivers for the development of new, efficient and flexible integrated purification processes. Currently, high-gradient magnetic separation as a direct capturing tool for protein purification suffers from the lack of suitable GMP-compliant separation equipment for industrial scale. As a solution for this bottleneck, we present a purification process for a monoclonal antibody directly from CHO cell culture by use of protein A-functionalized magnetic particles together with the first pilot-scale GMP-compliant ‘rotor-stator’ high-gradient magnetic separator. Five consecutive purification cycles were performed, achieving consistent yields of over 85% and purities of over 95%. Stable cell viabilities during the magnetic separation process enable integration of the device as an in situ product removal tool. A comparison with state-of-the-art protein A column-based purification processes reveals a 3-times higher process productivity per mL of applied resin and demonstrates the great potential of magnetic separation in downstream processing.

Preparation of Plasmid DNA by Alkaline Lysis with Sodium Dodecyl Sulfate: Maxipreps.

In recent years, with the advent of polymerase chain reaction and the development of highly efficient methods of cloning and DNA sequencing, the need to prepare large quantities of plasmid vectors and recombinants has greatly diminished. In consequence, this protocol, at one time in common use, has been largely replaced by faster and easier column-based purification methods. In this protocol, plasmid DNA is purified from the cleared bacterial lysate by centrifugation to equilibrium in CsCl gradients containing ethidium bromide. These gradients have an aesthetic quality that justifies the continued presentation of a protocol that in many other respects is an antique. The typical yield of high-copy-number plasmid vectors or of amplified low-copy-number vectors prepared by this method is ∼3-5 µg of DNA/mL of original bacterial culture. The yield of recombinant plasmids containing inserts of foreign DNA is usually slightly lower, depending on the size and nature of the cloned DNA fragment.

Hydroxamate column-based purification of zirconium-89 (89Zr) using an automated fluidic platform

Zirconium-89 (89Zr) is a long-lived (t1/2 = 78.4h) positron-emitting isotope that is useful for positron emission tomography (PET) based diagnostic imaging using radiolabeled antibodies. Hydroxamate resin columns are predominantly used for the purification of 89Zr from cyclotron bombarded natY targets dissolved in strong HCl. 89Zr is conventionally eluted from the resin in 1M oxalic acid (H2C2O4), a complexant that is conducive to follow-on binding of 89Zr through a transchelation process to the deferoxamine siderophore. In the present study, we determined that a lower concentration of H2C2O4 eluent (0.8M) is adequate to efficiently remove 89Zr from a column containing 100mg hydroxamate resin. As a result, less buffering agents are needed to be added to the 89Zr product fraction prior to labeling. A simple automated fluidic system prototype has been developed to perform the steps required for 89Zr purification using a hydroxamate resin column (column conditioning in HCl, Y target dissolution, dissolved target solution load onto column, column washes using HCl and water, and 89Zr elution). The system performance was evaluated using several cyclotron bombarded Y targets; 89Zr product fractions demonstrated excellent chemical recoveries from these targets, with 1.0mL product volumes yielding 89±2% of the column elution peak activity and 84±2% of 89Zr recovered from the target (at EOB). These results compare favorably with previously published 89Zr product volumes and yields, despite the lower concentration of H2C2O4 eluent employed. Transchelation of resulting 89Zr product fractions was performed to assess product quality. The effective specific activity (ESA) ranged between 44(7) and 109(22) TBq·mmole-1, while the bindable metals concentration, a metric introduced for assessing and comparing product purity, ranged between 43(7) and 115(27) nmole·g-1.

Rapid Quantification of the Latent Reservoir for HIV-1 Using a Viral Outgrowth Assay

HIV-1 persists in infected individuals in a stable pool of resting CD4+ T cells as a latent but replication-competent provirus. This latent reservoir is the major barrier to the eradication of HIV-1. Clinical trials are currently underway investigating the effects of latency-disrupting compounds on the persistence of the latent reservoir in infected individuals. To accurately assess the effects of such compounds, accurate assays to measure the frequency of latently infected cells are essential. The development of a simpler assay for the latent reservoir has been identified as a major AIDS research priority. We report here the development and validation of a rapid viral outgrowth assay that quantifies the frequency of cells that can release replication-competent virus following cellular activation. This new assay utilizes bead and column-based purification of resting CD4+ T cells from the peripheral blood of HIV-1 infected patients rather than cell sorting to obtain comparable resting CD4+ T cell purity. This new assay also utilizes the MOLT-4/CCR5 cell line for viral expansion, producing statistically comparable measurements of the frequency of latent HIV-1 infection. Finally, this new assay employs a novel quantitative RT-PCR specific for polyadenylated HIV-1 RNA for virus detection, which we demonstrate is a more sensitive and cost-effective method to detect HIV-1 replication than expensive commercial ELISA detection methods. The reductions in both labor and cost make this assay suitable for quantifying the frequency of latently infected cells in clinical trials of HIV-1 eradication strategies.

A One-Step Miniprep for the Isolation of Plasmid DNA and Lambda Phage Particles

Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs) have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments E.coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.

Abstract 4851: A single-tube protocol for next gen library construction increases complexity and simplifies parallel sample handling

Abstract With the advent of second-generation sequencing technologies, exome- and transcriptome- and whole genome sequencing are tools of choice in studying cancer to detect alterations in the genome correlating with tumorigenesis. It is essential to compare genomic alterations in cancer with matched normal DNA from the same individual largely due to incomplete knowledge of the “normal” variations. There have been concerted efforts for targeted sequencing of cancer-related exomes in a high throughput manner. However, the needs to process multiple samples simultaneously while maintaining the complexity of libraries have not been addressed adequately. Fragment library generation for single- and paired-end reads is typically performed by a series of enzymatic steps following gDNA shearing by ultrasonication. These may include end-repair, A-tailing, and ligation, each followed by bead- or column-based purification. The clean-ups are necessary to prevent enzyme activity carryover from one step to another. We have developed a single-tube method to take sheared, bead size-selected DNA through end-repair, A-tailing, and ligation without intermittent purifications. The A-tailing is done by a thermophilic polymerase at a temperature sufficient to denature the end-repair enzymes. Ligation is performed by adding ligase and adaptors after cooling the reaction. A bead- or column-based purification is then performed after the ligation reaction to produce template for amplification. With the removal of the clean-up steps, the workflow is simplified, allowing faster processing of samples with less hands-on time. We also reduce loss of sample associated with the purifications, increasing the complexity of the output library or enabling a lower sample input concentration. Our method is well suited for labs which are producing multiple libraries each week but have not scaled up to full automation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4851. doi:10.1158/1538-7445.AM2011-4851

Comparison of RNA extraction methods to augment the sensitivity for the differentiation of vesicular stomatitis virus Indiana1 and New Jersey

Two methods for the extraction of RNA of vesicular stomatitis virus (VSV) Indiana1 and New Jersey and their simultaneous amplification by one-step polymerase chain reaction using reverse transcriptase were evaluated. A guanidine-thiocyanate-based RNA extraction (Qiagen RNeasy Mini Kit, Qiagen, Valencia, CA ) followed by column-based purification coupled with one-step RT-PCR proved to be a simple, safe, practicable, and reliable tool for rapid, highly sensitive, and specific differential diagnosis of both types of VSV in cell lysate and spiked tissue samples as compared with the tri-phasic extraction method (Tri-reagent method). When RNA was extracted either from VSV cell culture stock or from VSV spiked bovine lymph nodes by using Qiagen RNeasy Mini Kit, the detection limit in the multiplex RT-PCR was as low as 0.505 to 2.84 TCID(50) for VSV-IND and VSV-NJ, respectively. The multiplex RT-PCR consistently detected VSV-IND and NJ RNA in as little as 0.1-1.0 fg of total RNA from spiked BHK-21 cell suspension when Qiagen RNeasy mini kit was used. The multiplex RT-PCR assay was capable of detecting both types of VSV in a one-step reaction tube. The minimum sensitivity of this assay in various experiments was 0.1683 TCID(50) (IND), 0.0946 TCID(50) (NJ), and 0.057 fg (IND and NJ) per 2 µl PCR sample, which is significantly more sensitive than reported previously (0.28-2.8 TCID50/1 µl). So the present study improved the sensitivity of previously reported multiplex RT-PCR for the detection and differentiation of VSV-IND and VSV-NJ in a single assay.

Quantitative Real-Time RT-PCR (qRT-PCR) of Zebrafish Transcripts: Optimization of RNA Extraction, Quality Control Considerations, and Data Analysis

The zebrafish (Danio rerio) has emerged as a popular model species. The rapid development of zebrafish embryos provides opportunities for investigation of genes essential for developmental processes, the human counterparts of which might be implicated in diseases. Understanding when and where genes are expressed can facilitate greater understanding of their function, and also allow the genes to be manipulated by gene knockdown in temporally and spatially specific manners. Quantitative real-time polymerase chain reaction (qRT-PCR) is widely applied in gene expression studies. This protocol presents techniques to optimize RNA isolation from zebrafish embryos; quality assessment and the use of multiple reference genes are also emphasized. The combined use of TRIzol extraction and column-based purification is strongly recommended, because the resulting RNA is of better quality than RNA isolated using either of those methods alone. The procedure can be performed in 2 d, with individual stages taking up to 15 h to complete.

Inhibitor-free DNA for real-time PCR analysis of synovial fluid from horses, cattle and pigs

The potential of five different commercial DNA isolation methods to remove real-time PCR inhibitors from the synovial fluid of horses, cattle and pigs was investigated. All kits with the exception of one included a silica column-based purification of the DNA. With the fifth kit, DNA purification is achieved by removing contaminating macromolecules by a desalting process. We used a recently developed method based on comparison of the real-time PCR signal of an artificial target incorporated into each PCR reaction in the presence of the isolated DNA from the sample, and in control samples containing water instead of isolated DNA. This was followed by statistical analysis of the data. Inhibition and subsequent reduction of the endpoint fluorescence in the real-time PCR reaction was encountered in many cases. Less frequently, the target copy number in the samples was underestimated. However, we found no experimental evidence of a negative influence of the reduced endpoint fluorescence signal on the detection limit of the real-time PCR assay. All kits tested were useful for analyzing pelleted synovial fluid from horses, cattle and pigs. When analyzing non-pelleted synovial fluid, three kits – two based on silica columns and one employing a desalting process – yielded inhibitor-free DNA for real-time PCR analysis.

High-throughput RFLP genotyping method for large genomes based on a chemiluminescent detection system

Genetic linkage mapping based on RFLPs is a valuable genomics tool for studying organisms with no genome sequence information. However, the generally used Southern hybridization method based on the radioisotope32P is not ideal for genotyping large mapping populations. We have overcome limitations of the alternative chemiluminescent detection system and developed a high-throughput RFLP genotyping method suitable for large-scale mapping studies of large genomes. Important elements in our process are PCR labeling of probes, complete removal of post-PCR unincorporated nucleotides via column-based purification methods, use of a 1:4 DIG-[11]-dUTP:dTTP ratio, and using a rocker instead of an orbital shaker during hybridization and post-hybridization processing of membranes. Using this method, we mapped the large genome of the homosporous fern speciesCeratopteris richardii by genotyping a mapping population of 513 doubled haploid line (DHL) progeny of a cross between two completely homozygous parental lines. Our genotyping method can robustly detect sub-picogram quantities of DNA fragments from a large number of samples and can be applied to linkage mapping studies of other organisms with large genomes.

424. Development of a Scalable Column Chromatography-Based Purification Process To Manufacure Empty Capsid-Free AAV Vectors

Recombinant adeno-associated virus (rAAV) has shown great promise as a DNA-based biopharmaceutical agent to treat serious human diseases. In conjunction with efforts in pre-clinical and clinical development, there is a significant need to develop and implement manufacturing processes capable of consistently providing vectors of optimal purity, potency and safety. We have previously reported the development and implementation of a fully scalable rAAV purification process that enabled us to manufacture AAV vectors at a scale to support our clinical development programs. However, like many other reported column-based purification processes, the vectors purified in our second generation purification method contained significant levels of empty capsids. In consideration of the possible deleterious effects of empty capsids, a product-related impurity, on vector efficacy and safety, we have developed a scalable method to separate empty capsids from vector particles. While this separation has been historically achieved using density gradient ultracentrifugation, such methods are not preferred for scalable, cost-effective vector manufacturing. Instead, we have developed a column chromatograpy-based technique to separate empty capsids from DNA containing vector particles. AAV vectors expressing human coagulation factor IX (AAV-hFIX16) were produced using helper virus-free triple transfection of HEK293 cells in roller bottles, and purified using anion exchange chromatography. This empty capsid-containing material was used as the starting point to screen various ion exchange resins to identify conditions that could differentiate between empty and full AAV2 particles. These studies indicated that the surface characteristics of the two particle types are very similar. However, identification of subtle, pH-dependent differences in binding to selected resins, in conjunction the selective use of chaotropic agents, allowed us to separate these two populations effectively. Our data indicated that more than 90% of the empty particles were removed from the purified vector using this newly developed column chromatography technique, with approximately 60% recovery of the DNA-containing vector particles. This new column step, in combination with the column-based scalable method that we previously reported, now enables us to efficiently manufacture empty capsid-free recombinant AAV using a fully scalable process.