Adeno-associated Virus Capsid Research Articles

Development of an optimized SEC method for characterization of genome DNA leakage from adeno-associated virus products.

Adeno-associated virus (AAV) vectors are widely used to deliver therapeutic transgenes due to their superior safety, relatively low immunogenicity, and ability to target diverse tissues. AAV gene therapy products are typically formulated as frozen liquid and stored below - 60 °C, and therefore are subjected to multiple freeze/thaw cycles during manufacturing and administration. Recent studies have shown that genome DNA leakage could be induced by freeze/thaw stress. DNA leakage from AAV capsids has been reported to potentially impact product stability, induce immune responses, and compromise product efficacy. Thus, further characterization to improve the understanding of genome DNA leakage is necessary for mitigating the risks associated with genome DNA leakage during AAV product development. In this work, we developed an optimized size-exclusion chromatography (SEC) method for quantifying the leakage of genome DNA across multiple different AAV serotypes and demonstrated satisfactory assay performance in sensitivity, precision, and linearity. Furthermore, we showed that this method could also be applied to quantifying additional quality attributes of AAV, including the percentage of full capsids and quantification of AAV dimers. By using this optimized SEC method, we demonstrated that significantly increased free DNA was observed with increasing freeze/thaw cycles or at a temperature approaching the onset temperature for genome DNA ejection, which was effectively mitigated by the addition of 1.5% w/v sucrose in the AAV formulation. Thus, this optimized SEC method can serve as an invaluable tool for AAV formulation, product, and process development in ensuring the quality and stability of AAV gene therapy products.

Adeno-associated virus 9 (AAV9) viral proteins VP1, VP2, and membrane-associated accessory protein (MAAP) differentially influence in vivo transgene expression.

Recombinant adeno-associated viruses (AAVs) are used extensively in clinical gene therapy for treating a range of tissues and pathologies in humans. In particular, AAV9 occupies a prominent position in central nervous system (CNS) gene therapy given its central role in ongoing clinical trials and an FDA-approved therapeutic. Despite its widespread use, recent studies have identified unique roles for the AAV capsid in in vivo transgene expression; for example, interior-facing capsid residues of AAV VP1 and VP2 modulate cellular transgene expression in vivo. The following experiments identified that the AAV9 MAAP protein exerts a significant influence on in vivo transgene expression. This finding could further explain how AAV can remain latent after infection in vivo. Together, these studies provide novel functional insights that highlight the importance of further understanding basic AAV biology.

Multimass Analysis of Adeno-Associated Virus Vectors by Orbitrap-Based Charge Detection Mass Spectrometry.

Adeno-associated virus (AAV) vectors have attracted significant attention as the main platform for gene therapy. To ensure the safety and efficacy of AAV vectors when used as gene therapy drugs, it is essential to assess their critical quality attributes (CQAs). These CQAs include the genome packaging status, the size of the genome encapsidated within the AAV capsid, and the stoichiometry of viral proteins (VPs) that constitute the AAV capsids. Analytical methods have been established for evaluating CQAs, such as analytical ultracentrifugation, capillary gel electrophoresis with laser-induced fluorescence detection, and capillary gel electrophoresis using sodium dodecyl sulfate with UV detection. Here, we present a multimass analysis of AAV vectors using orbitrap-based charge detection mass spectrometry (CDMS), a single-ion mass spectrometry. Orbitrap-based CDMS facilitates the quantitative evaluation of the genome packaging status based on the mass distribution of empty and full particles. Additionally, we established a novel method to analyze the encapsidated genome directly without pretreatment, such as protein digestion or heat treatment, and to estimate the stoichiometric variation of VP for the capsid based on the mass distribution constituted by the single peak corresponding to AAV particles. Orbitrap-based CDMS is a distinctive method that allows multiple mass characterizations of AAV vectors with a small sample volume of 20 μL for 1013 cp/mL in a short time (30 min), and it holds the potential to become a new standard method in the assessment of CQAs for AAV vectors.

Characterization of adeno-associated virus capsid proteins using denaturing size-exclusion chromatography coupled with mass spectrometry

Recombinant adeno-associated viruses (AAVs) are a highly effective platform for gene delivery for the treatment of many human diseases. Characterization of AAV viral protein attributes (VP), such as serotype identity, VP stoichiometry, and VP post-translational modifications, is essential to ensure product and process consistency. While size-exclusion chromatography (SEC) coupled with mass spectrometry (MS) is commonly used in the biopharmaceutical industry for analyzing protein therapeutics, its application to intact AAV VP components has not gained traction, presumably due to difficulties in achieving adequate resolution of VP(1−3) monomers. Herein, we describe the development of a denaturing SEC method and optimization of SEC parameters, including stationary phase pore size, column temperature, and mobile phase composition, to achieve effective chromatographic separation of VP(1−3). We demonstrate that an optimized dSEC-MS method featuring MS-compatible formic acid, can effectively separate VP(1−3) across AAV1, 2, 5, 6, 8, and 9 serotypes using a single column and mobile phase condition. A case study was included to showcase successful application of the dSEC-MS method in analyzing changes across different AAV production processes, yielding similar conclusions to an orthogonal approach, such as hydrophilic interaction chromatography (HILIC)- MS. Additionally, dSEC integrated with fluorescence (FLR) and ultraviolet (UV) detection can be used to semi-quantitatively identify both AAV DNA and VP components from empty and full AAV samples. Overall, this robust and MS-friendly methodological advancement could greatly streamline the development and analytical quality control processes for AAV-based gene therapies, providing a highly sensitive method for intact VP characterization.

Systemic gene therapy corrects the neurological phenotype in a mouse model of NGLY1 deficiency.

The cytoplasmic peptide:N-glycanase (NGLY1) is ubiquitously expressed and functions as a de-N-glycosylating enzyme that degrades misfolded N-glycosylated proteins. NGLY1 deficiency due to biallelic loss-of-function NGLY1 variants is an ultrarare autosomal recessive deglycosylation disorder with multisystemic involvement; the neurological manifestations represent the main disease burden. Currently, there is no treatment for this disease. To develop a gene therapy, we first characterized a tamoxifen-inducible Ngly1-knockout (iNgly1) C57BL/6J mouse model, which exhibited symptoms recapitulating human disease, including elevation of the biomarker GlcNAc-Asn, motor deficits, kyphosis, Purkinje cell loss, and gait abnormalities. We packaged a codon-optimized human NGLY1 transgene cassette into 2 adeno-associated virus (AAV) capsids, AAV9 and AAV.PHPeB. Systemic administration of the AAV.PHPeB vector to symptomatic iNgly1 mice corrected multiple disease features at 8 weeks after treatment. Furthermore, another cohort of AAV.PHPeB-treated iNgly1 mice were monitored over a year and showed near-complete normalization of the neurological aspects of the disease phenotype, demonstrating the durability of gene therapy. Our data suggested that brain-directed NGLY1 gene replacement via systemic delivery is a promising therapeutic strategy for NGLY1 deficiency. Although the superior CNS tropism of AAV.PHPeB vector does not translate to primates, emerging AAV capsids with enhanced primate CNS tropism will enable future translational studies.

AAVolve: Concatenated long-read deep sequencing enables whole capsid tracking during shuffled AAV library selection

Gene therapies using recombinant adeno-associated virus (AAV) vectors have demonstrated considerable clinical success in the treatment of genetic disorders. Improved vectors with favourable tropism profiles, decreased immunogenicity and enhanced manufacturability are poised to further improve the state of gene therapies. Such vectors can be identified through directed evolution, a process of subjecting a diverse capsid library to a selection pressure to identify individual variants with a desired trait. Currently, libraries that involve changes distributed throughout the AAV capsid coding region, such as DNA family shuffled libraries, are largely characterised using low-throughput Sanger sequencing of individual clones. However, improvements in long-read sequencing technologies have increased their applicability to capsid libraries and evaluation of the selection process. Here we explore the application of Oxford Nanopore Technologies refined by a concatemeric consensus method for initial library characterization and monitoring selection of a shuffled AAV capsid library. Furthermore, we present AAVolve, a bioinformatic pipeline for processing long-read data from AAV directed evolution experiments. Our approach allows high throughput characterisation of AAV capsids in a streamlined manner, facilitating deeper insights into library composition through multiple rounds of selection, and generalization through training of machine learning models.

Identification of a novel neutralization epitope in rhesus AAVs

Adeno-associated viruses (AAVs) are popular gene therapy delivery vectors, but their application can be limited by anti-vector immunity. Both preexisting neutralizing antibodies (NAbs) and post-administration NAbs can limit transgene expression and reduce the clinical utility of AAVs. The development of novel AAVs will advance our understanding of AAV immunity and may also have practical applications. In this study, we identified five novel AAV capsids from rhesus macaques. RhAAV4282 exhibited 91.4% capsid sequence similarity with AAV7 and showed similar tissue tropism with slightly diminished overall signal. Despite this sequence homology, RhAAV4282 and AAV7 showed limited cross-neutralization. We determined a cryo-EM structure of the RhAAV4282 capsid at 2.57Å resolution and identified a small segment within the hypervariable region IV, involving seven amino acids that formed a shortened external loop in RhAAV4282 compared with AAV7. We generated RhAAV4282 and AAV7 mutants that involved swaps of this region and showed that this region partially determined neutralization phenotype. We termed thisregion the hypervariable region IV neutralizing epitope (HRNE). Our data suggests that modification of the HRNE can lead to AAVs with altered neutralization profiles.

A Novel and Simplified Anion Exchange Flow-Through Polishing Approach for the Separation of Full From Empty Adeno-Associated Virus Capsids.

Adeno-associated viruses (AAV) are widely used viral vectors for in vivo gene therapy. The purification of AAV, particularly the separation of genome-containing from empty AAV capsids, is usually time-consuming and requires expensive equipment. In this study, we present a novel laboratory scale anion exchange flow-through polishing method designed to separate full and empty AAV. Once the appropriate conditions are defined, this method eliminates the need for a chromatography system. Determination of optimal polishing conditions using a chromatography system revealed that the divalent salt MgCl2 resulted in better separation of full and empty AAV than the monovalent salt NaCl. The efficacy of the method was demonstrated for three distinct AAV serotypes (AAV8, AAV5, and AAV2) on two different stationary phases: a membrane adsorber and a monolith, resulting in a 4- to 7.5-fold enrichment of full AAV particles. Moreover, the method was shown to preserve the AAV capsids' functional potency and structural integrity. Following the successful establishment of the flow-through polishing approach, it was adapted to a manual syringe-based system. Manual flow-through polishing using the monolith or membrane adsorber achieved 3.6- and 5.4-fold enrichment of full AAV, respectively. This study demonstrates the feasibility of separating full and empty AAV without complex linear or step gradient elution and the necessity of specialized equipment. Flow-through polishing provides a rapid and easy-to-perform platform for polishing multiple vector preparations, addressing a critical aspect in the research and development of novel gene therapies.

Expression-based selection identifies a microglia-tropic AAV capsid for direct and CSF routes of administration in mice.

Microglia are critical innate immune cells of the brain. In vivo targeting of microglia using gene-delivery systems is crucial for studying brain physiology and developing gene therapies for neurodegenerative diseases and other brain disorders such as NeuroAIDS. Historically, microglia have been extremely resistant to transduction by viral vectors, including adeno-associated virus (AAV) vectors. Recently, there has been some progress demonstrating the feasibility and potential of using AAV to transduce microglia after direct intraparenchymal vector injection. Data suggests that combining specific AAV capsids with microglia-specific gene expression cassettes to reduce neuron off-targeting will be key. However, no groups have developed AAV capsids for microglia transduction after intracerebroventricular (ICV) injection. The ICV route of administration has advantages such as increased brain biodistribution while avoiding issues related to systemic injection. Here, we performed an in vivo selection using an AAV peptide display library that enables recovery of capsids that mediate transgene expression in microglia. Using this approach, we identified a capsid, MC5, which mediated enhanced transduction of microglia after ICV injection compared to AAV9. Furthermore, MC5 enhanced both the efficiency (85%) and specificity (93%) of transduction compared to a recently described evolved AAV9 capsid for microglia targeting after direct injection into the brain parenchyma. Exploration of the use of MC5 in a mouse models of Alzheimer's disease revealed transduced microglia surrounding and within plaques. Overall, our results demonstrate that the MC5 capsid is a useful gene transfer tool to target microglia in vivo by direct and ICV routes of administration.

Structural Changes Likely Cause Chemical Differences between Empty and Full AAV Capsids.

Due to the success of adeno associated viruses (AAVs) in treating single-gene diseases, improved manufacturing technology is now needed to meet their demand. The largest challenge is creating a process to separate empty and full capsids. Patients received larger capsid doses than necessary due to the presence of empty capsids. By enabling the better separation of empty and full capsids, patients would receive the greatest therapeutic benefit with the least amount of virus capsids, thus limiting potential side effects from empty capsids. The two most common empty/full separation methods used in downstream processing are ultracentrifugation and anion exchange chromatography. Both processes have limitations, leading to a need for the identification of other structural differences that can be exploited to separate empty and full capsids. Here, we describe four possible theories of the structural changes that occur when AAV capsids envelop a genome. These theories include conformational changes occurring due to either the expansion or contraction of the capsid in the presence of nucleic acids, the constraining of the N-terminus into the five-fold pore when the genome is present, and the increased number of VP3 proteins in full capsids. These theories may reveal structural differences that can be exploited to separate full and empty capsids during manufacturing.

An engineered AAV targeting integrin alpha V beta 6 presents improved myotropism across species

Current adeno-associated virus (AAV) gene therapy using nature-derived AAVs is limited by non-optimal tissue targeting. In the treatment of muscular diseases (MD), high doses are often required but can lead to severe adverse effects. Here, we rationally design an AAV capsid that specifically targets skeletal muscle to lower treatment doses. We computationally integrate binding motifs of human integrin alphaV beta6, a skeletal muscle receptor, into a liver-detargeting capsid. Designed AAVs show higher productivity and superior muscle transduction compared to their parent. One variant, LICA1, demonstrates comparable muscle transduction to other myotropic AAVs with reduced liver targeting. LICA1’s myotropic properties are observed across species, including non-human primate. Consequently, LICA1, but not AAV9, effectively delivers therapeutic transgenes and improved muscle functionality in two mouse MD models (male mice) at a low dose (5E12 vg/kg). These results underline the potential of our design method for AAV engineering and LICA1 variant for MD gene therapy.

Human cell surface-AAV interactomes identify LRP6 as blood-brain barrier transcytosis receptor and immune cytokine IL3 as AAV9 binder

Adeno-associated viruses (AAVs) are foundational gene delivery tools for basic science and clinical therapeutics. However, lack of mechanistic insight, especially for engineered vectors created by directed evolution, can hamper their application. Here, we adapt an unbiased human cell microarray platform to determine the extracellular and cell surface interactomes of natural and engineered AAVs. We identify a naturally-evolved and serotype-specific interaction between the AAV9 capsid and human interleukin 3 (IL3), with possible roles in host immune modulation, as well as lab-evolved low-density lipoprotein receptor-related protein 6 (LRP6) interactions specific to engineered capsids with enhanced blood-brain barrier crossing in non-human primates after intravenous administration. The unbiased cell microarray screening approach also allows us to identify off-target tissue binding interactions of engineered brain-enriched AAV capsids that may inform vectors’ peripheral organ tropism and side effects. Our cryo-electron tomography and AlphaFold modeling of capsid-interactor complexes reveal LRP6 and IL3 binding sites. These results allow confident application of engineered AAVs in diverse organisms and unlock future target-informed engineering of improved viral and non-viral vectors for non-invasive therapeutic delivery to the brain.

AAV-DJ is superior to AAV9 for targeting brain and spinal cord, and de-targeting liver across multiple delivery routes in mice

Highly efficient adeno associated viruses (AAVs) targeting the central nervous system (CNS) are needed to deliver safe and effective therapies for inherited neurological disorders. The goal of this study was to compare the organ-specific transduction efficiencies of two AAV capsids across three different delivery routes. We compared AAV9-CBA-fLucYFP to AAV-DJ-CBA-fLucYFP using the following delivery routes in mice: intracerebroventricular (ICV) 1 × 1012 vg/kg, intrathecal (IT) 1 × 1012 vg/kg, and intravenous (IV) 1 × 1013 vg/kg body weight. Our evaluations revealed that following ICV and IT administrations, AAV-DJ demonstrated significantly increased vector genome (vg) uptake throughout the CNS as compared to AAV9. Through the IV route, AAV9 demonstrated significantly increased vg uptake in the CNS. However, significantly fewer vgs were detected in the off-target organs (kidney and liver) following administration of AAV-DJ using the IT and IV delivery routes as compared to AAV9. Distributions of vgs correlate well with transgene transcript levels, luciferase enzyme activities, and immunofluorescence detection of YFP. Overall, between the two vectors, AAV-DJ resulted in better targeting and expression in CNS tissues paired with de-targeting and reduced expression in liver and kidneys. Our findings support further examination of AAV-DJ as a gene therapy capsid for the treatment of neurological disorders.

Development of adeno-associated viral vectors targeting cardiac fibroblasts for efficient in vivo cardiac reprogramming

Overexpression of cardiac reprogramming factors, including GATA4, HAND2, TBX5, and MEF2C (GHT/M), can directly reprogram cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs). Adeno-associated virus (AAV) vectors are widely used clinically, and vectors targeting cardiomyocytes (CMs) have been extensively studied. However, safe and efficient AAV vectors targeting CFs for invivo cardiac reprogramming remain elusive. Therefore, we screened multiple AAV capsids and promoters to develop efficient and safe CF-targeting AAV vectors for invivo cardiac reprogramming. AAV-DJ capsids containing periostin promoter (AAV-DJ-Postn) strongly and specifically expressed transgenes in resident CFs in mice after myocardial infarction (MI). Lineage tracing revealed that AAV-DJ-Postn vectors expressing GHT/M reprogrammed CFs into iCMs, which was further increased 2-fold using activated MEF2C via the fusion of the powerful MYOD transactivation domain (M-TAD) with GHT (AAV-DJ-Postn-GHT/M-TAD). AAV-DJ-Postn-GHT/M-TAD injection improved cardiac function and reduced fibrosis after MI. Overall, we developed new AAV vectors that target CFs for cardiac reprogramming.

To 200,000 m/z and Beyond: Native Electron Capture Charge Reduction Mass Spectrometry Deconvolves Heterogeneous Signals in Large Biopharmaceutical Analytes.

Great progress has been made in the detection of large biomolecular analytes by native mass spectrometry; however, characterizing highly heterogeneous samples remains challenging due to the presence of many overlapping signals from complex ion distributions. Electron-capture charge reduction (ECCR), in which a protein cation captures free electrons without apparent dissociation, can separate overlapping signals by shifting the ions to lower charge states. The concomitant shift to higher m/z also facilitates the exploration of instrument upper m/z limits if large complexes are used. Here we perform native ECCR on the bacterial chaperonin GroEL and megadalton scale adeno-associated virus (AAV) capsid assemblies on a Q Exactive UHMR mass spectrometer. Charge reduction of AAV8 capsids by up to 90% pushes signals well above 100,000 m/z and enables charge state resolution and mean mass determination of these highly heterogeneous samples, even for capsids loaded with genetic cargo. With minor instrument modifications, the UHMR instrument can detect charge-reduced ion signals beyond 200,000 m/z. This work demonstrates the utility of ECCR for deconvolving heterogeneous signals in native mass spectrometry and presents the highest m/z signals ever recorded on an Orbitrap instrument, opening up the use of Orbitrap native mass spectrometry for heavier analytes than ever before.

Distinct chemical degradation pathways of AAV1 and AAV8 under thermal stress conditions revealed by analytical anion exchange chromatography and LC-MS-based peptide mapping

Adeno-associated virus (AAV)-based gene therapy is experiencing a rapid growth in the field of medicine and holds great promise in combating a wide range of human diseases. For successful development of AAV-based products, comprehensive thermal stability studies are often required to establish storage conditions and shelf life. However, as a relatively new modality, limited studies have been reported to elucidate the chemical degradation pathways of AAV products under thermal stress conditions. In this study, we first presented an intriguing difference in charge profile shift between thermally stressed AAV8 and AAV1 capsids when analyzed by anion exchange chromatography. Subsequently, a novel and robust peptide mapping protocol was developed and applied to elucidate the underlying chemical degradation pathways of thermally stressed AAV8 and AAV1. Compared to the conventional therapeutic proteins, the unique structure of AAV capsids also led to some key differences in how modifications at specific sites may impact the overall charge properties. Finally, despite the high sequency identity, the analysis revealed that the opposite charge profile shifts between thermally stressed AAV8 and AAV1 could be mainly attributed to a single modification unique to each serotype.

Predictive power of deleterious single amino acid changes to infer on AAV2 and AAV2-13 capsids fitness

Adeno-associated virus (AAV) is the most widely used vector for invivo gene transfer. A major limitation of capsid engineering is the incomplete understanding of the consequences of multiple amino acid variations on AAV capsid stability resulting in high frequency of non-viable capsids. In this context, the study of natural AAV variants can provide valuable insights into capsid regions that exhibit greater tolerance to mutations. Here, the characterization of AAV2 variants and the analysis of two public capsid libraries highlighted common features associated with deleterious mutations, suggesting that the impact of mutations on capsid viability is strictly dependent on their 3D location within the capsid structure. We developed a novel prediction method to infer the fitness of AAV2 variants containing multiple amino acid variations with 98% sensitivity, 98% accuracy, and 95% specificity. This novel approach might streamline the development of AAV vector libraries enriched in viable capsids, thus accelerating the identification of therapeutic candidates among engineered capsids.

Systematic multi-trait AAV capsid engineering for efficient gene delivery

Broadening gene therapy applications requires manufacturable vectors that efficiently transduce target cells in humans and preclinical models. Conventional selections of adeno-associated virus (AAV) capsid libraries are inefficient at searching the vast sequence space for the small fraction of vectors possessing multiple traits essential for clinical translation. Here, we present Fit4Function, a generalizable machine learning (ML) approach for systematically engineering multi-trait AAV capsids. By leveraging a capsid library that uniformly samples the manufacturable sequence space, reproducible screening data are generated to train accurate sequence-to-function models. Combining six models, we designed a multi-trait (liver-targeted, manufacturable) capsid library and validated 88% of library variants on all six predetermined criteria. Furthermore, the models, trained only on mouse in vivo and human in vitro Fit4Function data, accurately predicted AAV capsid variant biodistribution in macaque. Top candidates exhibited production yields comparable to AAV9, efficient murine liver transduction, up to 1000-fold greater human hepatocyte transduction, and increased enrichment relative to AAV9 in a screen for liver transduction in macaques. The Fit4Function strategy ultimately makes it possible to predict cross-species traits of peptide-modified AAV capsids and is a critical step toward assembling an ML atlas that predicts AAV capsid performance across dozens of traits.

Optimization of an adeno-associated viral vector for epidermal keratinocytes in vitro and in vivo

BackgroundLocal gene therapies, including in vivo genome editing, are highly anticipated for the treatment of genetic diseases in skin, especially the epidermis. While the adeno-associated virus (AAV) is a potent vector for in vivo gene delivery, the lack of efficient gene delivery methods has limited its clinical applications. ObjectiveTo optimize the AAV gene delivery system with higher gene delivery efficiency and specificity for epidermis and keratinocytes (KCs), using AAV capsid and promoter engineering technologies. MethodsAAV variants with mutations in residues reported to be critical to determine the tropism of AAV2 for KCs were generated by site-directed mutagenesis of AAVDJ. The infection efficiency and specificity for KCs of these variants were compared with those of previously reported AAVs considered to be suitable for gene delivery to KCs in vitro and in vivo. Additionally, we generated an epidermis-specific promoter using the most recent short-core promoter and compared its specificity with existing promoters. ResultsA novel AAVDJ variant capsid termed AAVDJK2 was superior to the existing AAVs in terms of gene transduction efficiency and specificity for epidermis and KCs in vitro and in vivo. A novel tissue-specific promoter, termed the K14 SCP3 promoter, was superior to the existing promoters in terms of gene transduction efficiency and specificity for KCs. ConclusionThe combination of the AAVDJK2 capsid and K14 SCP3 promoter improves gene delivery to epidermis in vivo and KCs in vitro. The novel AAV system may benefit experimental research and development of new epidermis-targeted gene therapies.

Membrane chromatography for AAV full capsid enrichment: Process development to scale up

The recent FDA approval of several adeno-associated virus (AAV)-based gene therapies is driving demand for AAV production. One of the biggest AAV manufacturing challenges is removing “empty” capsids, which do not contain the gene of interest. Anion exchange chromatography has emerged as the leading solution for scalable full capsid enrichment. Here we develop a process for the baseline separation of empty and full AAV capsids using anion exchange membrane chromatography. This process development approach utilized AAV serotypes 8 and 9 and traverses initial screening of separation conditions up to manufacturing-scale processes. Process development of a two-step elution was performed via response surface DoE, exploring conductivity and the length of the first elution step. The results from response surfaces were used to construct statistical models of the process operating space. These models provide optimal conditions for recovery and purity, both of which can exceed 70 %. Model predictions were then validated at small scale prior to scale-up. We present the results from our scale-up purification and show that purity and yield are consistent with the results obtained from the response surface model.