ACUVRA: Anion-Exchange Chromatography UV-Ratio Analysis-A QC-Friendly Method for Monitoring Adeno-Associated Virus Empty Capsid Content To Support Process Development and GMP Release Testing.

The genome content of adeno-associated virus (AAV) vectors is critical to the safety and potency of AAV-based gene therapy products. Empty capsids are considered a product-related impurity and a critical quality attribute (CQA) of the drug product, thus requiring characterization throughout the production process to demonstrate they are controlled to acceptable levels in the final drug product. Anion exchange chromatography has been used to achieve separation between empty and full capsids, but requires method development and gradient optimization for different serotypes and formulations. Here, we describe an alternative approach to quantitation that does not rely on achieving separation between empty and full capsids, but instead uses the well-established relationship between absorbance at UV A260/A280 and relation to DNA/protein content, in combination with anion-exchange chromatography to allow one to calculate the relative proportion of empty and full capsids in AAV samples from a single peak. We call this approach ACUVRA: Anion-exchange Chromatography UV-Ratio Analysis, and show the applicability of the method through a case study with recombinant AAV2 (rAAV2) process intermediates and drug substance. Method qualification and GMP validation in a quality control (QC) laboratory results show that ACUVRA is a fit-for-purpose method for process development support and characterization, while also being a QC-friendly option for GMP release testing at all stages of clinical development. Graphical abstract.

A qPCR Method for AAV Genome Titer with ddPCR-Level of Accuracy and Precision.

Recombinant adeno-associated virus (rAAV) is one of the main vectors used in gene therapy. An accurate genome titer is not only critical for clinical dosing, but also a prerequisite for many analytical assays for AAV product characterization. AAV genome titer is traditionally determined by qPCR; however, assay precision is not optimal despite extensive efforts. More recently, droplet digital PCR (ddPCR) emerged as a powerful alternative that offers excellent accuracy and precision. However, currently ddPCR is not as widely available as qPCR and operates at a lower throughput and a higher cost. In this paper, we introduce an improved qPCR method with two major optimizations: (1) using an AAV reference material as qPCR standard instead of plasmid DNA and (2) implementing a "digestion-free" method by adding 5% Tween 20 to standard and sample preparations. The new method has been extensively tested with AAV of different serotypes, purification status, and transgenes encapsidated and was found to be highly accurate, precise, and robust. This significantly improved and simplified assay can be easily adopted by researchers in the gene therapy field and further automated for high-throughput applications.

Assessment of the role of afucosylated glycoforms on the in vitro antibody-dependent phagocytosis activity of an antibody to Aß aggregates.

The terminal sugars of Fc glycans can influence the Fc-dependent biological activities of monoclonal antibody therapeutics. Afucosylated N-glycans have been shown to significantly alter binding to FcγRIIIa and affect antibody-dependent cell-mediated cytotoxicity (ADCC). Therefore, in order to maintain and ensure safety and efficacy for antibodies whose predominant mechanism of action (MOA) is ADCC, afucosylation is routinely monitored and controlled within appropriate limits. However, it is unclear how the composition and levels of afucosylated N-glycans can modulate the biological activities for a recombinant antibody whose target is not a cell surface receptor, as is the case with ADCC. The impact of different types and varying levels of enriched afucosylated N-glycan species on the in vitro bioactivities is assessed for an antibody whose target is aggregated amyloid beta (Aß). While either the presence of complex biantennary or high mannose afucosylated glycoforms significantly increased FcγRIIIa binding activity compared to fucosylated glycoforms, they did not similarly increase aggregated Aß uptake activity mediated by different effector cells. These experiments suggest that afucosylated N-glycans are not critical for the in vitro phagocytic activity of a recombinant antibody whose target is aggregated Aß and uses Fc effector function as part of its MOA.

Biolayer Interferometry-based FcγRIIa binding assay for a therapeutic antibody with strong effector function.

FcγRIIa receptor binding is part of the mechanism of action for many therapeutic antibodies. AlphaScreen® technology and Biolayer Interferometry (BLI) are often used to assess protein-protein interactions. Recently we demonstrated that the presence of aggregates in samples significantly increased binding potency values in AlphaScreen®-based FcRn binding assays, sometimes masking the loss of potency. Even bigger effect of aggregates was observed in an AlphaScreen®-based FcγRIIa binding assay for a monoclonal antibody with strong effector function. To resolve this issue a novel BLI-based FcγRIIa binding assay was developed and qualified. The assay measures association binding responses and calculates the binding potency of the samples relative to the standard using Parallel Line Analysis. The method overcomes interference of aggregates present in the samples, distinguishes different Fc glycosylation patterns, and is stability-indicating. It can be used for sample characterization, drug product release and stability testing.

Integration of Fluorescence Detection and Image-Based Automated Counting Increases Speed, Sensitivity, and Robustness of Plaque Assays.

Plaque assays are used to measure the infectious titer of viral samples. These assays are multi-day and low-throughput and may be subject to analyst variability from biased or subjective manual plaque counting. Typically, on day 1, cells are adhered to plates overnight. On day 2, cells are infected with virus. After 3 additional days, plaques are fixed, stained with a horseradish peroxidase (HRP)-conjugated antibody and a HRP substrate, and counted by eye. Manual-based visual counting of plaques is time-consuming and laborious and may be subject to variability between analysts. Also, the assay must proceed for several days to allow the plaques to increase to sufficiently large sizes for manual identification. Here, we integrate fluorescent detection and automated plaque counting to increase the sensitivity and speed of the assay. First, we stain plaques with a fluorescent-labeled antibody. Second, we implement a plate-based cell imager to perform non-biased, non-subjective plaque counting. The integration of these two technologies decreases the assay length by 40%, from 5 days to 3 days, because plaque size, plaque signal to noise, and manual visualization are no longer limiting. This optimized plaque assay is sensitive, fast, and robust and expands the throughput and usage of this method for measuring plaque formation.

Analytical Strategies for Quantification of Adeno-Associated Virus Empty Capsids to Support Process Development.

Recombinant adeno-associated virus (rAAV)-mediated gene therapy is a fast-evolving field in the biotechnology industry. One of the major challenges in developing a purification process for AAV gene therapy is establishing an effective yet scalable method to remove empty capsids, or viral vectors lacking the therapeutic gene, from full capsids-viral product containing the therapeutic sequence. Several analytical methods that can quantify the empty-to-full capsid ratio have been reported in the literature. However, as samples can vary widely in viral titer, buffer matrix, and the relative level of empty capsids, understanding the specifications and limitations of different analytical methods is critical to providing appropriate support to facilitate process development. In this study, we developed a novel anion-exchange high-performance liquid chromatography assay to determine the empty-to-full capsid ratio of rAAV samples. The newly developed method demonstrated good comparability with both the transmission electron microscopy and analytical ultracentrifugation methods used in empty-to-full capsid ratio quantification, while providing much higher assay throughput and reducing the minimum sample concentration requirement to 2.7E11 viral genomes/mL.

Monitoring long-term DNA storage via absolute copy number quantification by ddPCR.

Long-term storage of DNA is a routine practice in biomedical research, diagnostics and drug discovery. Periodic monitoring is important for early detection of changes in DNA quality and quantity. Existing methods include agarose gel, ultraviolet (UV) absorbance, fluorometric reading and qPCR. However, these methods are either limited by sensitivity or depend on DNA standards, which face the same storage challenges. In this paper, we tested the state-of-the-art droplet digital PCR (ddPCR) technology that can quantify the absolute DNA copy number with no need of a standard curve. We found that ddPCR was very accurate in determining the level of a plasmid DNA standard and was sensitive to DNA loss due to degradation or adsorption. With the ddPCR technology, we found a gradual process of DNA adsorption to several types of low binding tubes, which was unnoticed before. Although modest, adsorption significantly affected recovery of highly diluted DNA (<0.2 µg/mL), which could be rescued by addition of carrier DNA. In conclusion, this paper not only demonstrated that ddPCR is an ideal method for monitoring DNA storage, but also provided an effective approach to improving recovery of highly diluted DNA, which may have broad implications in assay development, diagnostics and forensic sciences.

A Digestion-free Method for Quantification of Residual Host Cell DNA in rAAV Gene Therapy Products.

Recombinant adeno-associated virus (rAAV) is a vector with increasing popularity in the field of gene therapy. Like other drug substances manufactured in cell lines, rAAV vectors are commonly contaminated with host cell DNA, and the levels must be carefully monitored. The current method for residual DNA quantification in rAAV was adapted from protein programs and required sample digestion by proteinase prior to qPCR analysis. While the method worked effectively, it was unclear if proteinase digestion was essential for releasing DNA from rAAV capsids and improving qPCR efficiency. In this study, we systematically investigated the role of each component and treatment with the goal to simplify and streamline the method. It was determined that the proteinase digestion step was dispensable, while the addition of Tween 20 to rAAV samples was essential for accurate quantification of residual DNA. Based on this finding, a digestion-free method has been established that requires only a one-step sample preparation-addition of Tween 20. The method has been tested extensively with an rAAV9-based drug substance and process intermediates and verified with other rAAV serotypes. This significantly simplified and faster assay can be easily automated for high-throughput applications.

Quantification of residual BHK DNA by a novel droplet digital PCR technology.

For drug substances manufactured in cell lines, host cell DNA is a common contaminant and its level must be carefully monitored. While residual DNA assays have been developed for many production cell lines, a robust assay is unavailable for baby hamster kidney (BHK) cells. The lack of genomics data of Syrian hamster, the origin of BHK cells, makes it challenging to design primers and probes for PCR-based methods. In this paper, we identified intracisternal A-particle (IAP) as an efficient PCR target for BHK DNA. PCR against IAP has been tested with conventional qPCR as well as with the recently developed ddPCR method, both of which demonstrated good efficiency with purified BHK DNA. However, the ddPCR-based method is less prone to matrix interference and is significantly more accurate than qPCR when testing complex samples, including multiple process intermediates. This study not only established a robust assay for the detection of residual BHK DNA, but also evaluated the capability of ddPCR technology for a new application.

Identification of adeno-associated virus capsid proteins using ZipChip CE/MS.

A simple and rapid identity test of adeno-associated virus (AAV) serotypes is important for supporting the AAV gene therapy development, as it relates to its efficacy and safety. The current mass spectrometry-based identity tests require extensive sample preparation steps, relatively large sample quantities and long analysis time. Herein, we describe a simple and novel microfluidic ZipChip CE/MS method used to characterize AAV capsid proteins. The three capsid proteins of AAV2 were separated and identified within 4 min using 5 nL of sample directly from a polysorbate-containing formulation buffer. This rapid method can be suitable to confirm AAV serotype identity.

Determination of Lentiviral Infectious Titer by a Novel Droplet Digital PCR Method.

Lentivirus is one of the best vehicles in delivering exogenous genes for therapeutics. Prior to application, it is very important to determine the infectious titer, which measures only mature virus capable of infecting target cells. Quantitative polymerase chain reaction (PCR) and fluorescence-activated cell sorting are commonly used for determination of infectious titer. This study introduces a new method based on Droplet Digital PCR (ddPCR), a recently developed PCR technology that quantifies the absolute amount of target DNA in the reaction. In this study, the dynamic range, Limit of Quantification (LOQ), and data acceptance criteria for ddPCR are defined against lentiviral sequence. ddPCR performance is also compared to established FACS and qPCR methods. This work not only demonstrates the feasibility of ddPCR in determining lentiviral infectious titer, but provides a detailed method that can be easily adapted by the scientific community.

A simple enzyme-substrate localized conjugation method to generate immobilized, functional glutathione S-transferase fusion protein columns for affinity enrichment.

Immobilized protein receptors and enzymes are tools for isolating or enriching ligands and substrates based on affinity. For example, glutathione S-transferase (GST) is fused to proteins as a tag for binding to its substrate glutathione (GSH) linked to solid supports. One issue with this approach is that high-affinity interactions between receptors and ligands require harsh elution conditions such as low pH, which can result in leached receptor. Another issue is the inherent nonspecific chemical conjugation of reactive groups such as N-hydroxysuccinimide (NHS) that couple lysines to solid supports; the nonspecificity of NHS may result in residue modifications near the binding site(s) of the receptor that can affect ligand specificity. In this study, a simple conjugation procedure is presented that overcomes these limitations and results in immobilized GST fusion proteins that are functional and specific. Here, the affinity of GST for GSH was used to generate an enzyme-substrate site-specific cross-linking reaction; GSH-Sepharose was preactivated with 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC) and then incubated Fc gamma receptor IIIa (FcγRIIIa)-GST. The immobilized FcγRIIIa-GST more specifically bound glycosylated immunoglobulin G1s (IgG1s) and was used to enrich nonfucosylated IgG1s from weaker binding species. This technique can be used when modifications of amino acids lead to changes in activity.

Effect of protein aggregates on characterization of FcRn binding of Fc-fusion therapeutics.

Recycling of antibodies and Fc containing therapeutic proteins by the neonatal Fc receptor (FcRn) is known to prolong their persistence in the bloodstream. Fusion of Fc fragment of IgG1 to other proteins is one of the strategies to improve their pharmacokinetic properties. Accurate measurement of Fc-FcRn binding provides information about the strength of this interaction, which in most cases correlates with serum half-life of the protein. It can also offer insight into functional integrity of Fc region. We investigated FcRn binding activity of a large set of Fc-fusion samples after thermal stress by the method based on AlphaScreen technology. An unexpected significant increase in FcR binding was found to correlate with formation of aggregates in these samples. Monomer purified from a thermally-stressed sample had normal FcRn binding, confirming that its Fc portion was intact. Experiments with aggregates spiked into a sample with low initial aggregation level, demonstrated strong correlation between the level of aggregates and FcRn binding. This correlation varied significantly in different methods. By introducing modifications to the assay format we were able to minimize the effects of aggregated species on FcRn binding, which should prevent masking functional changes of Fc-fusion protein. Biolayer interferometry (BLI) was used as an alternative method to measure FcRn binding. Both optimized AlphaScreen- and BLI-based assays were sensitive to structural changes in Fc portion of the molecule, such as oxidation of methionines 252 and 428, and therefore suitable for characterization of FcRn binding.

Development of a sensitive potency assay to measure the anti-proliferation effect of an anti-HER2 antibody.

For therapeutic antibodies that inhibit the growth of cancer cells, proliferation assays that measure cell number changes after the antibody treatment are often used to determine the potency of the antibody. Two of the most commonly used non-radioactive readout systems for proliferation assays, the ATP bioluminescence assay and the fluorescent dye Alamar Blue assay, were initially tested as potency assays an anti-HER2 antibody. Due to the slow growth of the target cells, these assays only produced less than 3-fold difference after 5 days of antibody treatment. BrdU incorporation-based proliferation assay, which differentiates proliferating cells from arrested cells, was developed, and showed superior sign-to-background ratio. Colorimetric, chemiluminescent, and DELFIA readouts were compared for BrdU incorporation assays, and DELFIA-based assay was further optimized using a Design of Experiment (DoE) approach. The final DELFIA-based BrdU incorporation assay demonstrated superior signal-to-background ratio, robustness, accuracy, and precision, and represented significant improvement over traditional proliferation assays.

Development and qualification of a high sensitivity, high throughput Q-PCR assay for quantitation of residual host cell DNA in purification process intermediate and drug substance samples.

Methods of high sensitivity, accuracy and throughput are needed for quantitation of low level residual host cell DNA in purification process intermediates and drug substances of therapeutic proteins. In this study, we designed primer/probe sets targeting repetitive Alu repeats or Alu-equivalent sequences in the human, Chinese hamster and murine genomes. When used in quantitative polymerase chain reactions (Q-PCRs), these primer/probe sets showed high species specificity and gave significantly higher sensitivity compared to those targeting the low copy number GAPDH gene. This allowed for detection of residual host cell DNA of much lower concentrations and, for some samples, eliminated the need for DNA extraction. By combining the high sensitivity Alu Q-PCR with high throughput automated DNA extraction using an automated MagMAX magnetic particle processor, we successfully developed and qualified a highly accurate, specific, sensitive and efficient method for the quantitation of residual host cell DNA in process intermediates and drug substances of multiple therapeutic proteins purified from cells of multiple species. Compared to the previous method using manual DNA extraction and primer/probe sets targeting the GAPDH gene, this new method increased our DNA extraction throughput by over sevenfold, and lowered the lower limit of quantitation by up to eightfold.

Advances in the assessment and control of the effector functions of therapeutic antibodies.

The Fc (crystallizable fragment) region of therapeutic antibodies can have an important role in their safety and efficacy. Although much is known about the structure-activity relationship of antibodies and the factors that influence Fc effector functions, a process has not yet been defined to clearly delineate how Fc functionality should be assessed and controlled during antibody development and manufacturing. In this article, we summarize the current knowledge of antibody Fc functionality, provide a strategy for assessing the effector functions of different classes of therapeutic antibodies (including Fc fusion proteins) and propose a path for routine testing and controls for manufacturers of antibody products.

2'-C-Methyl branched pyrimidine ribonucleoside analogues: potent inhibitors of RNA virus replication.

RNA viruses are the agents of numerous widespread and often severe diseases. Their unique RNA-dependent RNA polymerase (RDRP) is essential for replication and, thus, constitutes a valid target for the development of selective chemotherapeutic agents. In this regard, we have investigated sugar-modified ribonucleoside analogues as potential inhibitors of the RDRP. Title compounds retain 'natural' pyrimidine bases, but possess a beta-methyl substituent at the 2'-position of the D- or L-ribose moiety. Evaluation against a broad range of RNA viruses, either single-stranded positive (ssRNA+), single-stranded negative (ssRNA-) or double-stranded (dsRNA), revealed potent activities for D-2'-C-methyl-cytidine and -uridine against ssRNA+, and dsRNA viruses. None of the L-enantiomers were active. Moreover, the 5'-triphosphates of the active D-enantiomers were found to inhibit the bovine virus diarrhoea virus polymerase. Thus, the 2'-methyl branching of natural pyrimidine ribonucleosides transforms physiological molecules into potent, broad-spectrum antiviral agents that merit further development.