Formulation Buffer Research Articles

High-Throughput Single-Particle Characterization of Aggregation Pathways and the Effects of Inhibitors for Large (Megadalton) Protein Oligomers.

Protein aggregation is involved in many human diseases, but characterizing the sizes and shapes of intermediate oligomers (∼10-100 nm) that are important to the formation of macroscale aggregates like amyloid fibrils is a significant analytical challenge. Here, charge detection mass spectrometry (CDMS) is used to characterize individual conformational states of bovine serum albumin oligomers with up to ∼225 molecules (15 MDa). Elongated, partially folded, and globular conformational families for each oligomer can be readily distinguished based on the extent of charging. The abundances of individual conformers vary with changes in the monomer concentration or by adding aggregation inhibitors, such as SDS, heparin, or MgCl2. These results show the potential of CDMS for investigating intermediate oligomers in protein aggregation processes that are important for understanding aggregate formation and inhibition mechanisms and could accelerate formulation buffer development to prevent the aggregation of biotherapeutics.

Ionizable Drugs Enable Intracellular Delivery of Co-Formulated siRNA.

Targeting complementary pathways in diseases such as cancer can be achieved with co-delivery of small interfering ribonucleic acid (siRNA) and small molecule drugs; however, current formulation strategies are typically limited to one, but not both. Here, ionizable small molecule drugs and siRNA are co-formulatedin drug-rich nanoparticles. Ionizable analogs of the selective estrogen receptor degrader fulvestrant self-assemble into colloidal drug aggregates and cause endosomal disruption, allowing co-delivery of siRNA against a non-druggable target. siRNA is encapsulated in lipid-stabilized, drug-rich colloidal nanoparticles where the ionizable lipid used in conventional lipid nanoparticles is replaced with an ionizable fulvestrant analog. The selection of an appropriate phospholipid and formulation buffer enables endocytosis and potent reporter gene knockdown in cancer cells. Importantly, siRNA targeting cyclin E1 is effectively delivered to drug-resistant breast cancer cells, demonstrating the utility of this approach. This strategy opens the possibility of using ionizable drugs to co-deliver RNA and ultimately improve therapeutic outcomes.

Long term investigation of formulation buffers to mitigate stability issues of conjugated critical reagents

Stability of conjugated critical reagents supporting ligand binding assays to enable biotherapeutic drug development is a universal concern. Formulation buffer employed for long-term cold storage may be key to mitigate protein aggregation issues. We investigated biophysical and functional attributes of murine mAb and human multispecific drug labeled with biotin, ruthenium, and Alexa fluor 647 frozen at −80 °C in PBS or a protein storage buffer for 3–15 months. Aggregation was observed at 4 months in mAb A-Ru (11.2%) and -Alexa (10%) in PBS followed by precipitation and reduced biological binding at 15 months. Increased aggregation in drug Ru (11.7%, 6 months) and Alexa (6.9%, 15 months) were noted but without impact on performance. There were no observations with biotin labeled reagents.

Near UV and Visible Light Photodegradation in Solid Formulations: Generation of Carbon Dioxide Radical Anions from Citrate Buffer and Fe(III).

Near UV and visible light photodegradation can target therapeutic proteins during manufacturing and storage. While the underlying photodegradation pathways are frequently not well-understood, one important aspect of consideration is the formulation, specifically the formulation buffer. Citrate is a common buffer for biopharmaceutical formulations, which can complex with transition metals, such as Fe(III). In an aqueous solution, the exposure of such complexes to light leads to the formation of the carbon dioxide radical anion (•CO2-), a powerful reductant. However, few studies have characterized such processes in solid formulations. Here, we show that solid citrate formulations containing Fe(III) lead to the photochemical formation of •CO2-, identified through DMPO spin trapping and HPLC-MS/MS analysis. Factors such as buffers, the availability of oxygen, excipients, and manufacturing processes of solid formulations were evaluated for their effect on the formation of •CO2- and other radicals such as •OH.

Safety and immunogenicity of the live-attenuated hRVFV-4s vaccine against Rift Valley fever in healthy adults: a dose-escalation, placebo-controlled, first-in-human, phase 1 randomised clinical trial

Rift Valley fever virus, a pathogen to ruminants, camelids, and humans, is an emerging mosquito-borne bunyavirus currently endemic to Africa and the Arabian Peninsula. Although animals are primarily infected via mosquito bites, humans mainly become infected following contact with infected tissues or fluids of infected animals. There is an urgent need for adequate countermeasures, especially for humans, because effective therapeutics or vaccines are not yet available. Here we assessed the safety, tolerability, and immunogenicity of a next-generation, four-segmented, live-attenuated vaccine candidate, referred to as hRVFV-4s, in humans. A first-in-human, single-centre, randomised, double-blind, placebo-controlled trial was done in Belgium in which a single dose of hRVFV-4s was administered to healthy volunteers aged 18-45 years. Participants were randomly assigned using an interactive web response system. The study population encompassed 75 participants naive to Rift Valley fever virus infection, divided over three dosage groups (cohorts) of 25 participants each. All participants were followed up until 6 months. Using a staggered dose escalating approach, 20 individuals of each cohort were injected in the deltoid muscle of the non-dominant arm with either 104 (low dose), 105 (medium dose), or 106 (high dose) of 50% tissue culture infectious dose of hRVFV-4s as based on animal data, and five individuals per cohort received formulation buffer as a placebo. Primary outcome measures in the intention-to-treat population were adverse events and tolerability. Secondary outcome measures were vaccine-induced viraemia, vaccine virus shedding, Rift Valley fever virus nucleocapsid antibody responses (with ELISA), and neutralising antibody titres. Furthermore, exploratory objectives included the assessment of cellular immune responses by ELISpot. The trial was registered with the EU Clinical Trials Register, 2022-501460-17-00. Between August and December, 2022, all 75 participants were vaccinated. No serious adverse events or vaccine-related severe adverse events were reported. Pain at the injection site (51 [85%] of 60 participants) was most frequently reported as solicited local adverse event, and headache (28 [47%] of 60) and fatigue (28 [47%] of 60) as solicited systemic adverse events in the active group. No vaccine virus RNA was detected in any of the blood, saliva, urine, or semen samples. Rift Valley fever virus nucleocapsid antibody responses were detected in most participants who were vaccinated with hRVFV-4s (43 [72%] of 60 on day 14) irrespective of the administered dose. In contrast, a clear dose-response relationship was observed for neutralising antibodies on day 28 with four (20%) of 20 participants responding in the low-dose group, 13 (65%) of 20 responding in the medium-dose group, and all participants (20 [100%] of 20) responding in the high-dose group. Consistent with the antibody responses, cellular immune responses against the nucleocapsid protein were detected in all dose groups, whereas a more dose-dependent response was observed for the Gn and Gc surface glycoproteins. Neutralising antibody titres declined over time, whereas nucleocapsid antibody responses remained relatively stable for at least 6 months. The hRVFV-4s vaccine showed a high safety profile and excellent tolerability across all tested dose regimens, eliciting robust immune responses, particularly with the high-dose administration. The findings strongly support further clinical development of this candidate vaccine for human use. The Coalition for Epidemic Preparedness Innovations with support from the EU Horizon 2020 programme.

It is time to reconsider the use of naturally-occurring endotoxins in endotoxin recovery studies: Part 2 of a BioPhorum harmonized endotoxin recovery study

The evaluation of Naturally Occurring Endotoxins (NOEs) for Low Endotoxin Recovery (LER) studies has been a topic in the industry and regulatory agencies have been hesitant to endorse NOE use in LER studies over purified Lipopolysaccharide (LPS) standards such as Control Standard Endotoxin (CSE) or Reference Standard Endotoxin (RSE). In a recent study involving 11 BioPhorum member companies across 13 sites, NOEs prepared in high and low nutrient conditions were evaluated in two common monoclonal antibody buffer formulations: 10 mM Sodium Citrate, 0.05 % Polysorbate 80, pH 6.0 and 20 mM Histidine, 0.05 % Polysorbate 80, pH 6.0. 12 g-negative bacterial isolates were used to prepare NOE analytes, which were spiked into the formulation buffers. Additionally, the NOEs were spiked into Limulus Amebocyte Lysate (LAL) reagent water as controls and purified LPS into the citrate/polysorbate buffer as the LER control. Results showed the average of three runs per organism was >50 % recovery, at the conclusion of the 7-day period, regardless of nutrient culture preparation conditions. Furthermore, purified LPS controls became undetectable (<50 % recovery) in the citrate/polysorbate buffer, highlighting the presence of LER. These findings highlight the potential value of using NOEs from relevant manufacturing facilities to assess overall risk when purified LPS recovery is insufficient.

Respiratory syncytial virus vaccination during pregnancy for improving infant outcomes.

Respiratory syncytial virus vaccination during pregnancy for improving infant outcomes.

Development of a fluorescence-based excipient screening for improved stability and shelf-life of recombinant chitin deacetylase

Chitin deacetylase (CDA) modifies chitin into chitosan by removing acetyl groups, but its inherent instability poses a challenge for successful crystallisation. Despite limited successes in crystallizing CDAs, prior attempts with recombinant chitin deacetylase (BaCDA) failed due to poor stability. To address this, we propose an enzyme buffer formulation as a cost-effective strategy to enhance stability, prolong shelf life, and increase the likelihood of crystallisation. Utilizing the high-throughput screening technique FTSA, we developed a screening method correlating BaCDA stability with its activity. The optimised formulation comprises 50 mM Tris-HCl buffer pH 7, 1 M NaCl, 20 % glycerol, and 1 mM Mg2+ as excipients. This formulation significantly improves BaCDA's thermostability (140.47 % increase) and enzyme activity (2.9-fold enhancement). BaCDA remains stable in the formulated buffer at −20 °C and −80 °C for 30 days and at 4 °C for 15 days. The current study has designed a high-throughput screening method approach to assess the stability of CDA enzyme formulations. The results of this study could contribute to the exploration of formulation elements that enhance the structural stability of CDA, thereby facilitating investigations into the enzyme's structure-function relationships.

Analysis of the impact of pluronic acid on the thermal stability and infectivity of AAV6.2FF

BackgroundThe advancement of AAV vectors into clinical testing has accelerated rapidly over the past two decades. While many of the AAV vectors being utilized in clinical trials are derived from natural serotypes, engineered serotypes are progressing toward clinical translation due to their enhanced tissue tropism and immune evasive properties. However, novel AAV vectors require formulation and stability testing to determine optimal storage conditions prior to their use in a clinical setting.ResultsHere, we evaluated the thermal stability of AAV6.2FF, a rationally engineered capsid with strong tropism for lung and muscle, in two different buffer formulations; phosphate buffered saline (PBS), or PBS supplemented with 0.001% non-ionic surfactant Pluronic F68 (PF-68). Aliquots of AAV6.2FF vector encoding the firefly luciferase reporter gene (AAV6.2FF-ffLuc) were incubated at temperatures ranging from -20°C to 55°C for varying periods of time and the impact on infectivity and particle integrity evaluated. Additionally, the impact of several rounds of freeze-thaw treatments on the infectivity of AAV6.2FF was investigated. Vector infectivity was measured by quantifying firefly luciferase expression in HEK 293 cells and AAV particle integrity was measured by qPCR quantification of encapsidated viral DNA.ConclusionsOur data demonstrate that formulating AAV6.2FF in PBS containing 0.001% PF-68 leads to increased stability and particle integrity at temperatures between -20℃ to 21℃ and protection against the destructive effects of freeze-thaw. Finally, AAV6.2FF-GFP formulated in PBS supplemented with 0.001% PF-68 displayed higher transduction efficiency in vivo in murine lung epithelial cells following intranasal administration than vector buffered in PBS alone further demonstrating the beneficial properties of PF-68.

Quantification of Residual Water in Pharmaceutical Frozen Solutions Via 1H Solid-State NMR

Freezing is essential for the stability of biological drug substances and products, particularly in frozen solution formulations and during the primary drying of lyophilized preparations. However, the unfrozen segment within the frozen matrix can alter solute concentration, ionic strength, and stabilizer crystallization, posing risks of increased biophysical instability and faster chemical degradation. While quantifying the unfrozen water content is important for designing stable biopharmaceuticals, there is a lack of analytical techniques for in situ quantitative measurements. In this study, we introduce a 1H magic angle spinning NMR technique to identify the freezing point (Tice) and quantify mobile water content in frozen biologics, applying this method to analyze the freezing of a commercial high-concentration drug product, Dupixent®. Our results demonstrate that water freezing is influenced by buffer salt properties and formulation composition, including the presence of sugar cryoprotectants and protein concentration. Additionally, the 1H chemical shift can probe pH in the unfrozen phase, potentially predicting the microenvironmental acidity in the frozen state. Our proposed methodology provides fresh insights into the analysis of freeze-concentrated solutions, enhancing our understanding of the stability of frozen and lyophilized biopharmaceuticals.

Optimizing lentiviral vector formulation conditions for efficient ex vivo transduction of primary human T cells in chimeric antigen receptor T-cell manufacturing

Background aimsChimeric antigen receptor (CAR) T-cell products are commonly generated using lentiviral vector (LV) transduction. Optimal final formulation buffer (FFB) supporting LV stability during cryostorage is crucial for cost-effective manufacturing. MethodsTo identify the ideal LV FFB composition for ex vivo CAR-T production, primary human T cells were transduced with vesicular stomatitis virus G-protein (VSV-G) -pseudotyped LVs (encoding a reporter gene or an anti-CD19-CAR). The formulations included phosphate-buffered saline (PBS), HEPES, or X-VIVOTM 15, and stabilizing excipients. The functional and viral particle titers and vector copy number were measured after LV cryopreservation and up to 24 h post-thaw incubation. CAR-Ts were produced with LVs in selected FFBs, and the resulting cells were characterized. ResultsPost-cryopreservation, HEPES-based FFBs provided higher LV functional titers than PBS and X-VIVOTM 15, and 10% trehalose-20 mM MgCl2 improved LV transduction efficiency in PBS and 50 mM HEPES. Thawed vectors remained stable at +4°C, while a ≤ 25% median decrease in the functional titer occurred during 24 h at room temperature. Tested excipients did not enhance LV post-thaw stability. CAR-Ts produced using LVs cryopreserved in 10% trehalose- or sucrose-20 mM MgCl2 in 50 mM HEPES showed comparable transduction rates, cell yield, viability, phenotype, and in vitro functionality. ConclusionA buffer consisting of 10% trehalose-20 mM MgCl2 in 50 mM HEPES provided a feasible FFB to cryopreserve a VSV-G -pseudotyped LV for CAR-T-cell production. The LVs remained relatively stable for at least 24 h post-thaw, even at ambient temperatures. This study provides insights into process development, showing LV formulation data generated using the relevant target cell type for CAR-T-cell manufacturing.

Introduction of a fatty acid chain modification to prolong circulatory half-life of a radioligand towards glucose-dependent insulinotropic polypeptide receptor

BackgroundThe beneficial role of glucose-dependent insulinotropic polypeptide receptor (GIPR) in weight control and maintaining glucose levels has led to the development of several multi-agonistic peptide drug candidates, targeting GIPR and glucagon like peptide 1 receptor (GLP1R) and/or the glucagon receptor (GCGR). The in vivo quantification of target occupancy by these drugs would accelerate the development of new drug candidates. The aim of this study was to evaluate a novel peptide (GIP1234), based on previously reported ligand DOTA-GIP-C803, modified with a fatty acid moiety to prolong its blood circulation. It would allow higher target tissue exposure and consequently improved peptide uptake as well as in vivo PET imaging and quantification of GIPR occupancy by novel drugs of interest. MethodA 40 amino acid residue peptide (GIP1234) was synthesized based on DOTA-GIP-C803, in turn based on the sequences of endogenous GIP and Exendin-4 with specific amino acid modifications to obtain GIPR selectivity. A palmitoyl fatty acid chain was furthermore added at Lys14 via a glutamic acid linker to prolong its blood circulation time by the interaction with albumin. GIP1234 was conjugated with a DOTA chelator at the C-terminal cysteine residue to achieve 68Ga radiolabeling. The resulting PET probe, [68Ga]Ga-DOTA-GIP1234 was evaluated for receptor binding specificity and selectivity using HEK293 cells transfected with human GIPR, GLP1R, or GCGR. Blocking experiments with tirzepatide (2 μM) were conducted using huGIPR HEK293 cells to investigate binding specificity. Ex vivo and in vivo organ distribution of [68Ga]Ga-DOTA-GIP1234 was studied in rats and a pig in comparison to [68Ga]Ga-DOTA-C803-GIP. Binding of [68Ga]Ga-DOTA-GIP1234 to albumin was assessed in situ using polyacrylamide gel electrophoresis (PAGE). The stability was tested in formulation buffer and rat blood plasma. Results[68Ga]Ga-DOTA-GIP1234 was synthesized with non-decay corrected radiochemical yield of 88 ± 3.7 % and radiochemical purity of 97.8 ± 0.8 %. The molar activity for the radiotracer was 8.1 ± 1.1 MBq/nmol. [68Ga]Ga-DOTA-GIP1234 was stable and maintained affinity to huGIPR HEK293 cells (dissociation constant (Kd) = 40 ± 12.5 nM). The binding of [68Ga]Ga-DOTA-GIP1234 to huGCGR and huGLP1R cells was insignificant. Pre-incubation of huGIPR HEK293 cell sections with tirzepatide resulted in the decrease of [68Ga]Ga-DOTA-GIP1234 binding by close to 90 %. [68Ga]Ga-DOTA-GIP1234 displayed slow blood clearance in pigs with SUV = 3.5 after 60 min. Blood retention of the tracer in rat was 2-fold higher than that of [68Ga]Ga-DOTA-C803-GIP. [68Ga]Ga-DOTA-GIP1234 also demonstrated strong liver uptake in both pig and rat combined with decreased renal excretion. The concentration dependent binding of [68Ga]Ga-DOTA-GIP1234 to albumin was confirmed in situ by PAGE. Conclusion[68Ga]Ga-DOTA-GIP1234 demonstrated nanomolar affinity and selectivity for huGIPR in vitro. Addition of a fatty acid moiety prolonged blood circulation time and tissue exposure in both rat and pig in vivo. However, the liver uptake was also increased which may make PET imaging of abdominal tissues such as pancreas challenging. The investigation of the influence of fatty acid moiety on the biological performance of the peptide ligand paved the way for further rational design of GIPR ligand analogues with improved characteristics.

The NMR signature of maltose-based glycation in full-length proteins

Reducing sugars can spontaneously react with free amines in protein side chains leading to posttranslational modifications (PTMs) called glycation. In contrast to glycosylation, glycation is a non-enzymatic modification with consequences on the overall charge, solubility, aggregation susceptibility and functionality of a protein. Glycation is a critical quality attribute of therapeutic monoclonal antibodies. In addition to glucose, also disaccharides like maltose can form glycation products. We present here a detailed NMR analysis of the Amadori product formed between proteins and maltose. For better comparison, data collection was done under denaturing conditions using 7 M urea-d4 in D2O. The here presented correlation patterns serve as a signature and can be used to identify maltose-based glycation in any protein that can be denatured. In addition to the model protein BSA, which can be readily glycated, we present data of the biotherapeutic abatacept containing maltose in its formulation buffer. With this contribution, we demonstrate that NMR spectroscopy is an independent method for detecting maltose-based glycation, that is suited for cross-validation with other methods.

Aggregation of human plasma and of human blood induced in vitro by filgrastim originator product; effect of PEGylation

We herein report that filgrastim product Neupogen® and the filgrastim formulation buffer induced aggregate formation when mixed in vitro with human plasma, and formation of large membranous erythrocyte aggregates when mixed with human blood, similar to the aggregation induced by pegfilgrastim and by pegfilgrastim buffer [T. Arvinte, E. Poirier, N. Ersayin, G. Darpin, A. Cudd, J. Dowd, S. Brokx, Aggregation of human plasma and of human blood induced in vitro by pegfilgrastim originator formulation buffer and pegfilgrastim products, Eur. J. Pharmaceut. Biopharmaceut. (2023), doi: 10.1016/j.ejpb.2023.10.019]. The data identify the filgrastim buffer (which is practically the same in filgrastim and pegfilgrastim products) as the main driver of human plasma and blood aggregation. Kinetic experiments showed differences in the extent of plasma aggregation induced by a filgrastim product manufactured in EU and one manufactured in USA. Human donor variability in the plasma aggregation induced by filgrastim was observed. To study the effect of PEGylation of the filgrastim protein on plasma aggregation we compared filgrastim (Neupogen®) with pegfilgrastim (Neulasta®) solutions at the same protein concentration. These data show that PEGylation has a beneficial effect in inhibiting to an extent plasma aggregation. Interestingly, 20 kDa polyethylene glycol in the filgrastim buffer induced more plasma aggregation compared to the buffer, similar to the aggregation induced by pegfilgrastim. For intravenous infusion filgrastim solutions (300 µg/ml, vials only) may be diluted in 5 % dextrose from a concentration of 300 µg/ml to 5 µg/ml. Aggregation of human plasma was also induced by filgrastim solutions diluted in 5 % dextrose to 50 µg/ml, 15 µg/ml and 5 µg/ml filgrastim, as well as by the filgrastim buffer similarly diluted in 5 % dextrose (1:6, 1:20 and 1:60 dilution). These data show that filgrastim solutions used for intravenous administration in patients induce human plasma aggregation in vitro. Such aggregation phenomena may be related to known infusion side effects of filgrastim therapy.

Aggregation of human plasma and of human blood induced in vitro by pegfilgrastim originator formulation buffer and pegfilgrastim products

PEGylated recombinant human granulocyte colony stimulating factor (pegfilgrastim) is used clinically to reduce the incidence and duration of severe neutropenia in patients who have received chemotherapy treatment. Pegfilgrastim products are administered by subcutaneous injection. We herein report that solutions of pegfilgrastim originator product Neulasta®, of a biosimilar product candidate, and also of the pegfilgrastim originator formulation buffer, induced aggregate formation when mixed in vitro with human plasma, and formation of large membranous aggregated structures when mixed with human blood. Human donor variability in the plasma aggregation induced by pegfilgrastim products was observed. In all donors less aggregation occurred in plasma mixtures with the biosimilar pegfilgrastim product candidate compared to the originator products. Instantaneous aggregation of erythrocytes and formation of large membranous aggregated structures of erythrocytes occurred in mixtures of human blood with pegfilgrastim buffer or pegfilgrastim products. The formation of the large membranous aggregated structures likely involved fusion of erythrocyte membranes; erythrocyte membrane fusion events were observed. Pegfilgrastim proteins in the products accelerated the formation of irreversible erythrocyte aggregated structures. Pegfilgrastim originator formulation buffer (10 mM Na-acetate pH 4.0, 274 mM sorbitol, 0.004 % polysorbate 20) was identified as the main driver of the plasma and erythrocyte aggregation. Lipoprotein aggregation at low pH in the presence of sorbitol and erythrocyte membrane fusion induced by the lipoprotein aggregates, are proposed as the main mechanisms for the formation of plasma and blood aggregates. Such aggregation phenomena may also occur during pegfilgrastim clinical use and may be related to known side effects and individual variability in the efficacy of pegfilgrastim therapy.

B-221 Quantitation of DNA in Antibody-based and RNA-based Drugs by Quantitative Polymerase Chain Reaction (qPCR)

Abstract Background Current FDA regulation defined acceptance level of 10 ng per dose of residual DNA in drug products poses great challenges for accurate quantification of residual DNA, particularly for the products used in high doses or formulated in complex matrixes. The purpose of this study is to develop and qualify a qPCR method for the quantification of Chinese Hamster Ovary (CHO) DNA in antibody-based drugs and for quantitation of human DNA in RNA-based drugs. Methods An 8-point standard curve consisting of 0.005, 0.05, 0.5, 5, 50, 500, 1000 and 5000 pg CHO DNA and a 5-point standard curve consisting of 0.3, 3, 30, 300 and 3000 pg human DNA was generated by serial dilutions. The antibody sample (300 µg) was spiked with 2.5, 10, and 50 pg CHO DNA. The RNA sample (1.6 µg) was spiked with 0.3, 3 and 30 pg human DNA. The samples were analyzed by qPCR using a Real-Time PCR instrument. Commercial PCR test kits (TaqMan™ Probe and TaqMan™ Universal Master Mix), and sequence specific forward and reverse primers were used. Results For CHO DNA in antibody product, the linearity slope and R2 are −3.5 and 1.00, respectively, which met system suitability criteria. The accuracy by spiked recovery of 2.5 pg, 10 pg and 50 pg CHO DNA in 300 µg antibody, 150 pg CHO DNA in formulation buffer, and 150 pg CHO DNA in 1000 pg human DNA are 70%, 76%, 86%, 93% and 95%, respectively. The precision of the assay for 2.5 pg, 10 pg, and 50 pg of CHO DNA spiked in 300 µg antibody are 6%, 7% and 2%, respectively. The QL of the assay is 2.5 pg CHO DNA per 300 µg of antibody product.For human DNA in RNA product, the linearity slope and R2 are −3.3 and 1.00, respectively, which met system suitability criteria. The accuracy by spiked recovery of 0.3 pg, 3 pg, and 30 pg human DNA in 1.6 µg RNA, 3 pg human DNA in formulation buffer, 1.5 pg human DNA in 150 pg CHO DNA are 113%, 104%, 102%, 97% and 88%, respectively. The precision of the assay for 0.3 pg, 3 pg and 30 pg human DNA spiked in 1.6 µg RNA are 6%, 5% and 2%, respectively. The QL of the assay is 0.3 pg human DNA per 1.6 µg of RNA product. Conclusion Two qPCR methods were developed to quantify residual DNA in drug products. One method is specific to quantitation of low levels of CHO DNA in antibody product. Another method is specific to quantitation of low levels of human DNA in RNA product.

Safety and biodistribution of XC001 (encoberminogene rezmadenovec) gene therapy in rats: a potential therapy for cardiovascular diseases.

Adenovirus-mediated gene therapy holds promise for the treatment of cardiovascular diseases such as refractory angina. However, potential concerns around immunogenicity and vector dissemination from the target injected tissue require evaluation. This study was undertaken to evaluate the safety and biodistribution of XC001, a replication-deficient adenovirus serotype 5 vector expressing multiple isoforms of human vascular endothelial growth factor (VEGF), following direct administration into normal rat myocardium. Animals received the buffer formulation or increasing doses of XC001 (1 × 107, 2.5 × 108 or 2.5 × 109 viral particles). Based on in-life parameters (general health, body weights, clinical pathology, serum cardiac troponin I, plasma VEGF, and gross necropsy), there were no findings of clinical concern. On Day 8, intramyocardial administration of XC001 was associated with dose-related, left ventricular myocardial inflammation at injection sites, resolving by Day 30. XC001 DNA was not detected in blood at any time but was present at Day 8 around the site of injection and to a much lesser extent in the spleen, liver, and lungs, persisting at low levels in the heart and spleen until at least Day 91. These findings demonstrate that intramyocardial injection of XC001 is supported for use in human studies.

Optimization of Drug-Linker to Enable Long-term Storage of Antibody-Drug Conjugate for Subcutaneous Dosing.

To facilitate subcutaneous dosing, biotherapeutics need to exhibit properties that enable high-concentration formulation and long-term stability in the formulation buffer. For antibody-drug conjugates (ADCs), the introduction of drug-linkers can lead to increased hydrophobicity and higher levels of aggregation, which are both detrimental to the properties required for subcutaneous dosing. Herein we show how the physicochemical properties of ADCs could be controlled through the drug-linker chemistry in combination with prodrug chemistry of the payload, and how optimization of these combinations could afford ADCs with significantly improved solution stability. Key to achieving this optimization is the use of an accelerated stress test performed in a minimal formulation buffer.

Preclinical Pharmacokinetic Profile of Topical Ophthalmic and Intravenous Delivery of QLS-101, a Novel ATP-Sensitive Potassium Channel Opening Ocular Hypotensive Agent.

Purpose: To evaluate the pharmacokinetic profiles of the ocular hypotensive agent QLS-101, a novel ATP-sensitive potassium channel opening prodrug, and its active moiety levcromakalim, following topical ophthalmic and intravenous dosing of normotensive rabbits and dogs. Methods: Dutch belted rabbits (n = 85) and beagle dogs (n = 32) were dosed with QLS-101 (0.16-3.2 mg/eye/dose) or formulation buffer for 28 days. Pharmacokinetic profiles of QLS-101 and levcromakalim were evaluated in ocular tissues and blood by LC-MS/MS. Tolerability was assessed by clinical and ophthalmic examinations. Maximum systemic tolerated dose was evaluated in beagle dogs (n = 2) following intravenous bolus administrations of QLS-101 (0.05 to 5 mg/kg). Results: Plasma analysis following topical dosing of QLS-101 (0.8-3.2 mg/eye/dose) for 28 days indicated an elimination half-life (T1/2) of 5.50-8.82 h and a corresponding time (Tmax) range of 2-12 h in rabbits, and a T1/2 of 3.32-6.18 h with a Tmax range of 1-2 h in dogs. Maximum tissue concentration (Cmax) values ranged from 54.8-540 (day 1) to 50.5-777 ng/mL (day 28) in rabbits, and 36.5-166 (day 1) to 47.0-147 ng/mL (day 28) in dogs. Levcromakalim plasma T1/2 and Tmax were similar to QLS-101, while Cmax was consistently lower. Topical ophthalmic delivery of QLS-101 was well tolerated in both species, with sporadic mild ocular hyperemia noted in the group treated with the highest concentration (3.2 mg/eye/dose). Following topical ophthalmic dosing, QLS-101 and levcromakalim were found primarily in the cornea, sclera, and conjunctiva. Maximum tolerated dose was determined to be 3 mg/kg. Conclusions: QLS-101 was converted to its active moiety levcromakalim and showed characteristic absorption, distribution, and safety profiles of a well-tolerated prodrug.

AAV analysis by sedimentation velocity analytical ultracentrifugation: beyond empty and full capsids

The recent surge of therapeutic interest in recombinant adeno-associated viral (AAV) vectors for targeted DNA delivery has brought analytical ultracentrifugation (AUC) into the spotlight. A major concern during formulation of AAV therapeutics is purity of the active species (DNA-containing capsid, or "filled capsids"). Insertion of DNA into AAV is not a highly efficient process; thus, a significant amount of empty and partial/intermediate AAV molecules may exist. Recent guidance from the FDA includes limiting the presence of empty AAV capsids and other impurities to reduce immunotoxicity. While chromatographic techniques (SEC, SEC-MALS, AEX) are often used for empty and full capsid quantitation due to the ease of accessibility and familiarity among most biochemists, the resolution and sensitivity attained by sedimentation velocity (SV-AUC) in the formulation buffer and purification buffers is unmatched. Approaches for using SV-AUC to determine the empty-to-full capsid ratio have already been discussed by others; however, in this report, we focus on the importance of characterizing other impurities, such as free DNA, partially filled capsids, and aggregates that are recognized as species of concern for immunotoxicity. We also demonstrate the usefulness of applying multiple analyses (e.g., c(s), g(s*), WDA) in confirming the presence of and determining the hydrodynamic parameters of these various species.