Analytical characterization of full, intermediate, and empty AAV capsids.

Manufacturing of recombinant adeno-associated virus (AAV) vectors produces three types of capsids: full, intermediate, and empty. While there are different opinions about the impact of intermediate and empty capsids on safety and efficacy of AAV products, they are generally considered impurities because they are not the intended fully intact vector product. The presence of these impurities could impact product efficacy due to potential competition with fully packaged AAVs for cellular transduction, as well as have potential implications to patient safety due to increased capsid load during dosing. To determine the impact of intermediate capsids on potency, an AAV preparation was separated into fractions enriched for full, intermediate, or empty capsids. Using a matrix of in vitro (infectivity, gene expression, biological activity) and in vivo potency assays to determine potency as a function of capsid content, our results indicate that while intermediate capsids contribute to the vector genome titer of the product and are equally as infectious as full capsids, they do not contribute to the potency of the AAV product. This study confirms the criticality of reducing and controlling the level of intermediate capsids to ensure a more efficacious AAV product.

A novel dual-plasmid platform provides scalable transfection yielding improved productivity and packaging across multiple AAV serotypes and genomes.

Transient transfection of mammalian cells using plasmid DNA is a standard method to produce adeno-associated virus (AAV) vectors allowing for flexible and scalable manufacture. Typically, three plasmids are used to encode the necessary components to facilitate vector production; however, a dual-plasmid system, termed pDG, was introduced over 2 decades ago demonstrating two components could be combined resulting in comparable productivity to triple transfection. We have developed a novel dual-plasmid system, pOXB, with an alternative arrangement of sequences that results in significantly increased AAV vector productivity and percentage of full capsids packaged in comparison to the pDG dual design and triple transfection. Here, we demonstrate the reproducibility of these findings across seven recombinant AAV genomes and multiple capsid serotypes as well as the scalability of the pOXB dual-plasmid transfection at 50-L bioreactor scale. Purified drug substance showed a consistent product quality profile in line with triple-transfected vectors, except for a substantial improvement in intact genomes packaged using the pOXB dual- transfection system. Furthermore, pOXB dual- and triple-transfection-based vectors performed consistently in vivo. The pOXB dual plasmid represents an innovation in AAV manufacturing resulting in significant process gains while maintaining the flexibility of a transient transfection platform.

Strategy for Generating Sequence-Defined Aptamer Reagent Sets for Detecting Protein Contaminants in Biotherapeutics.

Biologic drugs are typically manufactured in mammalian host cells, and it is critical from a drug safety and efficacy perspective to detect and remove host cell proteins (HCPs) during production. This is currently achieved with sets of polyclonal antibodies (pAbs), but these suffer from critical shortcomings because their composition is inherently undefined, and they cannot detect nonimmunogenic HCPs. In this work, we report a high-throughput screening and array-based binding characterization strategy that we employed to generate a set of aptamers that overcomes these limitations to achieve sensitive, broad-spectrum detection of HCPs from the widely used Chinese hamster ovary (CHO) cell line. We identified a set of 32 DNA aptamers that achieve better sensitivity than a commercial pAb reagent set and can detect a comparable number of HCPs over a broad range of isoelectric points and sizes. Importantly, these aptamers detect multiple contaminants that are known to be responsible for therapeutic antibody degradation and toxicity in patients. Because HCP aptamer reagents are sequence-defined and chemically synthesized, we believe they may enable safer production of biologic drugs, and this strategy should be broadly applicable for the generation of HCP detection reagents for other cell lines.

Comprehensive analysis of host cell impurities in monoclonal antibodies with improved sensitivity by capillary zone electrophoresis mass spectrometry.

Four methods were compared for analysis of host-cell protein (HCP) impurities in a recombinant mAb. First, CZE-MS/MS was used to analyze the digest of an HCP sample following extraction of the mAb with proteins A and L affinity columns; 220 protein groups and 976 peptides were identified from the depleted HCP digest. Second, a nanoACQUITY UltraPerformance LCH system was also used to analyze the depleted HCP digest; 34 protein groups and 53 peptides from 50 ng of the depleted HCP digest and 290 protein groups and 1011 peptides were identified from 1 µg of the depleted HCP digest. Third, 185 protein groups and 709 peptides were identified by CZE-MS/MS from the HCP digest without depletion. Fourth, a strong cation exchange SPE was coupled to CZE-ESI-MS/MS using online pH gradient elution for analysis of the HCP digest without depletion. A series of five pH bumps were applied to elute peptides from the strong cation exchange monolith followed by analysis using CZE coupled to a Q Exactive HF mass spectrometer; 230 protein groups and 796 peptides were identified from the HCP digest without depletion.

Quantitative analysis of the supernatant from host and transfected CHO cells using iTRAQ 8-plex technique.

We employed UPLC-MS/MS with iTRAQ 8-plex labeling to quantitatively analyze the supernatant produced by two Chinese hamster ovary (CHO) cell lines (CHO K1SV and CHO CAT-S). In each case, the supernatant from the host and three transfected clones were analyzed at days 5, 7, and 10 of culture. A total of eight iTRAQ 8-plex experiments were performed. For each cell line, the overlap of supernatant protein identifications between transfected clones is over 60%. Over 70% of the supernatant proteins in the CHO K1SV host cell line are present in the CHO CAT-S cell line. For the CHO K1SV cell line, the overlap in supernatant protein identifications between the host cell line and the transfected clones is >59%. For the CHO CAT-S cell line, the overlap between supernatant protein identifications for the transfected clone and host cell is >45%. These differences in the supernatant protein identifications between transfected clones in each cell line and between the two host cell lines are not significant. We used cluster analysis to characterize the change in supernatant protein expression as a function of cell culture time. Roughly <60% of the supernatant proteins show significant change across the three time points (ratio >1.3 or <0.7). We also used cluster analysis to compare changes in supernatant protein expression between the host and three transfected clones at each time point. Greater than 65% of the common proteins in the CHO K1SV cell line supernatant and over 54% in the CHO CAT-S cell line supernatant show no significant expression difference between host and the three transfected clones. Data are available via ProteomeXchange with identifier PXD003462. Biotechnol. Bioeng. 2016;113: 2140-2148. © 2016 Wiley Periodicals, Inc.

Rapid Identification of Biotherapeutics with Label-Free Raman Spectroscopy.

Product identification is a critical and required analysis for biotheraputics. In addition to regulatory requirements for identity testing on final drug products, in-process identity testing is implemented to reduce business risks associated with fill operations and can also be used as a tool against counterfeiting. Biotherapeutics, in particular monoclonal antibodies, represent a challenging cohort for identity determination because of their similarity in chemical structure. Traditional methods used for product identification can be time and labor intensive, creating a need for quick, inexpensive and reliable methods of drug identification. Here, driven by its molecular-specific and nonperturbative nature, we present Raman spectroscopy as an alternate analytical tool for identity testing. By exploiting subtle differences in vibrational modes of the biologics, we have developed partial least-squares-discriminant analysis derived decision algorithms that offer excellent differentiation capability using spontaneous Raman spectra as well as label-free plasmon-enhanced Raman spectra. Coupled with the robustness to spurious correlations due to its high information content, our results highlight the potential of Raman spectroscopy as a powerful method for rapid, on-site biotherapeutic product identification.

Capillary zone electrophoresis tandem mass spectrometry detects low concentration host cell impurities in monoclonal antibodies.

We have evaluated CZE-ESI-MS/MS for detection of trace amounts of host cell protein impurities in recombinant therapeutics. Compared to previously published procedures, we have optimized the buffer pH used in the formation of a pH junction to increase injection volume. We also prepared a 5-point calibration curve by spiking 12 standard proteins into a solution of a human mAb. A custom CZE-MS/MS system was used to analyze the tryptic digest of this mixture without depletion of the antibody. CZE generated a ∼70-min separation window (∼90-min total analysis duration) and ∼300-peak capacity. We also analyzed the sample using ultra-performance LC-MS/MS. CZE-MS/MS generated approximately five times higher base peak intensity and more peptide identifications for low-level spiked proteins. Both methods detected all proteins spiked at ∼100 ppm level with respect to the antibody.

Absolute quantitation of host cell proteins in recombinant human monoclonal antibodies with an automated CZE-ESI-MS/MS system.

We report the first use of CZE for absolute characterization of host cell proteins (HCPs) in recombinant human monoclonal antibodies. An electrokinetically pumped nanoelectrospray interface was used to couple CZE with a tandem mass spectrometer. Three isotopic-labeled peptides (LSFDKDAMVAR, VDIVENQAMDTR, and LVSDEMVVELIEK) were synthesized by direct incorporation of an isotope-labeled lysine or arginine. The heavy-labeled peptides were spiked in the HCP digests at known concentrations. After CZE-ESI-MS/MS analysis, the peaks of native and isotopic-labeled peptides were extracted with mass tolerance ≤ 5 ppm from the electropherograms, and the ratios of peak area between native and isotopic-labeled peptides pairs were calculated. Calibration curves (the ratios of peak area versus spiked peptide amount) with R(2) values of 0.999, 0.997, and 0.999 were obtained for the three HCP peptides, and the absolute amounts of the three proteins present were determined to be at the picomole level in a 20 µg sample of digested HCPs. The target proteins were present at the 7-30 ppt level in the purified HCP samples.

A rapid cIEF-ESI-MS/MS method for host cell protein analysis of a recombinant human monoclonal antibody.

A rapid and reproducible system that couples capillary isoelectric focusing to a high-resolution mass spectrometer was developed for on-line analysis and identification of protein digests. Magnetic microsphere-based immobilized trypsin was used for protein digestion to reduce the digestion time to 10 min, with a total analysis time of 4h. A three-protein-mixture (myoglobin, BSA, cytochrome c) with a molarity ratio of 1:10:50 was successfully digested and identified. This system was also used to analyze host cell protein impurities in a recombinant humanized monoclonal antibody product in which the sample was product-depleted using affinity capture on protein A/protein L columns prior to analysis. A database search identified 37 host cell proteins with peptide and protein identity probability greater than 0.9.

Evaluation of a synthetic peptide as a replacement for the recombinant fusion protein of respiratory syncytial virus in a potency ELISA.

This report describes the development of a potency ELISA using a peptide derived from the motavizumab binding epitope of respiratory syncytial virus (RSV) F-protein. Motavizumab is an antibody therapeutic studied for the prevention of RSV disease. It binds to the RSV glycoprotein F (F-protein), blocking the ability of RSV to fuse with target cells. This binding is the basis for a potency ELISA, however, due to inefficient F-protein production, development of an alternative ligand for the potency ELISA was investigated. A series of synthetic peptides spanning the motavizumab epitope on F-protein were evaluated for motavizumab binding activity. A 26-mer peptide was identified with desirable motavizumab binding kinetics, as shown by ELISA and surface plasmon resonance. The peptide corresponds to a portion of the motavizumab binding domain on the F-protein, and is referred to as F-peptide. The binding of motavizumab to the F-peptide is used in a new motavizumab potency ELISA, which was shown to be robust and statistically comparable to the F-protein ELISA. In addition, based on a qualitative observation, this new ELISA may be able to detect motavizumab degradation with greater sensitivity compared to the F-protein ELISA.

Forensic dental training in the dental school curriculum.

This article reviews the literature regarding forensic education in the dental school curriculum and describes an exercise in forensic identification of victims of a mass casualty. Radiographs were made of dentate human cadavers in the gross anatomy laboratory at the Southern Illinois School of Dental Medicine. The jaws were then removed to provide "wet specimens" for the exercise. Several restorations were performed on the cadaver teeth, after which radiographs of the dissected jaws were made. One author wrote up mock dental records for each of the victims. These records included the first set or "premortem" radiographs. Students participating in the exercise were provided with a plane crash scenario, the dental records of the passengers on the manifest, the dissected jaws, and the second set or "postmortem" radiographs. Students were expected to form three teams. The first two teams evaluated the ante-mortem and postmortem dental records. The third team compared the ante-mortem and postmortem records to arrive at identification. The purpose of the exercise was twofold. It introduced dental students to forensic dentistry and emphasized the need for complete and accurate record keeping in the dental office. Several factors lessened the realism of the exercise and made it difficult to reproduce in the future. These included the uniformity of the dental records and the destruction of cadaver material following the exercise.