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.

Analytical Quality by Design, Life Cycle Management, and Method Control.

Analytical methods are utilized throughout the biopharmaceutical and vaccines industries to conduct research and development, and to help control manufacturing inputs and outputs. These analytical methods should continuously provide quality data to support decisions while managing the remaining of risk and uncertainty. Analytical quality by design (AQbD) can provide a systematic framework to achieve a continuously validated, robust assay as well as life cycle management. AQbD is rooted in ICH guidelines Q8 and Q9 that were translated to the analytical space through several white papers as well as upcoming USP 1220 and ICH Q14. In this white paper, we expand on the previously published concepts of AQbD by providing additional context for implementation in relation to ICH Q14. Using illustrative examples, we describe the AQbD workflow, its relation to traditional approaches, and potential pathways for ongoing, real-time verification. We will also discuss challenges with respect to implementation and regulatory strategies.

NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods.

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

Rapid quantification of tyrosine sulfation in therapeutic proteins.

Protein tyrosine sulfation (Tyr-O-SO3) is a common post-translational modification (PTM), which is important for protein function. Absolute quantitation of Tyr-O-SO3 in recombinant therapeutic proteins has been challenging. We report here an MRM method used for absolute quantitation of Tyr-O-SO3 in the hydrolysate of a recombinant Fc-fusion protein. Quantitation is achieved by monitoring the sum of two transitions: the loss of carboxylic acid from tyrosine sulfate (major transition) and sulfate group from tyrosine sulfate sodium salt. The method exhibits a good sensitivity with a limit of quantitation of 1.4 ng/mL, linearity over three orders of magnitude, good repeatability, precision and accuracy.

A high-throughput capillary isoelectric focusing immunoassay for fingerprinting protein sialylation.

The serum half-life, biological activity, and solubility of many recombinant glycoproteins depend on their sialylation. Monitoring glycoprotein sialylation during cell culture manufacturing is, therefore, critical to ensure product efficacy and safety. Here a high-throughput method for semi-quantitative fingerprinting of glycoprotein sialylation using capillary isoelectric focusing immunoassay on NanoPro (Protein Simple) platform was developed. The method was specific, sensitive, precise, and robust. It could analyze 2 µL of crude cell culture samples without protein purification, and could automatically analyze from 8 samples in 4 h to 96 samples in 14 h without analyst supervision. Furthermore, its capability to detect various changes in sialylation fingerprints during cell culture manufacturing process was indispensable to ensure process robustness and consistency. Moreover, the changes in the sialylation fingerprints analyzed by this method showed strong correlations with intact mass analysis using liquid chromatography and mass spectrometry.

Residual metals cause variability in methionine oxidation measurements in protein pharmaceuticals using LC-UV/MS peptide mapping.

Methionine oxidation has been demonstrated to play an important role in protein stability in vitro and in vivo. It may also cause changes in biological activity and immunogenicity profile of therapeutic proteins. Therefore, it is critical to monitor methionine oxidation in biopharmaceuticals during process and formulation development, as well as long-term stability studies. A common analytical method for methionine oxidation determination is peptide mapping analysis of protein enzymatic digests using UV detection with or without mass spectrometric detection. The quantitation of oxidation is performed based on the UV or extracted ion chromatographic peak areas of the oxidized and non-oxidized peptides. This method was found to be susceptible to significant variability over long-term use. Major factors leading to this variability included presence of low levels of metal ions, especially iron, in the digestion buffer, chromatographic column, LC injector, and other sample contact surfaces. Careful control of metal ion levels generally leads to less variability and long-term consistency of peptide mapping methods for oxidation determination.

Metabolomics profiling of cell culture media leading to the identification of riboflavin photosensitized degradation of tryptophan causing slow growth in cell culture.

As more protein biopharmaceuticals are produced using mammalian cell culture techniques, it becomes increasingly important for the biopharmaceutical industry to have tools to characterize the cell culture media and evaluate its impact on the cell culture performance. Exposure of the cell culture media to light, temperature stress, or adventitious introduction of low-level organisms during preparation can lead to the generation of chemical degradants or metabolites of the media components, which are potentially detrimental to the cell culture process. In this work, we applied a liquid chromatography-mass spectrometry based metabolomics methodology for the investigation of a media lot used for a mammalian cell culture process that had resulted in low growth rate and failure to meet required viable cell density (VCD). The study led to the observation of increased levels of tryptophan oxidation products and a riboflavin degradant, lumichrome, in the malfunctioning media lot, relative to working media lots. A compound found 7-fold higher in the working media lots appeared to be tetrahydropentoxyline, a condensation product of glucose and tryptophan. A second compound found at an over 50-fold higher level in the malfunctioning media lot with a proposed molecular formula of C(21)H(17)N(3)O(3) from high-resolution mass spectrometry (HRMS) analysis remains unknown, although it is confirmed to be a degradant of tryptophan in the media. A study of the cell culture media performed under stress conditions using fluorescent light and heat showed that the media powder was highly resistant to light-induced degradation, while solution media could be easily degraded after brief light exposure. It is therefore suspected that inadvertent exposure of the media to light during preparation and storage has resulted in the poor performance of the media causing the low growth and VCD in the cell culture process.