Oligonucleotide mapping via mass spectrometry to enable comprehensive primary structure characterization of an mRNA vaccine against SARS-CoV-2.

Oligonucleotide mapping via liquid chromatography with UV detection coupled to tandem mass spectrometry (LC-UV-MS/MS) was recently developed to support development of Comirnaty, the world's first commercial mRNA vaccine which immunizes against the SARS-CoV-2 virus. Analogous to peptide mapping of therapeutic protein modalities, oligonucleotide mapping described here provides direct primary structure characterization of mRNA, through enzymatic digestion, accurate mass determinations, and optimized collisionally-induced fragmentation. Sample preparation for oligonucleotide mapping is a rapid, one-pot, one-enzyme digestion. The digest is analyzed via LC-MS/MS with an extended gradient and resulting data analysis employs semi-automated software. In a single method, oligonucleotide mapping readouts include a highly reproducible and completely annotated UV chromatogram with 100% maximum sequence coverage, and a microheterogeneity assessment of 5' terminus capping and 3' terminus poly(A)-tail length. Oligonucleotide mapping was pivotal to ensure the quality, safety, and efficacy of mRNA vaccines by providing: confirmation of construct identity and primary structure and assessment of product comparability following manufacturing process changes. More broadly, this technique may be used to directly interrogate the primary structure of RNA molecules in general.

Characterization of BNT162b2 mRNA to Evaluate Risk of Off-Target Antigen Translation.

mRNA vaccines have been established as a safe and effective modality, thanks in large part to the expedited development and approval of COVID-19 vaccines. In addition to the active, full-length mRNA transcript, mRNA fragment species can be present as a byproduct of the cell-free transcription manufacturing process or due to mRNA hydrolysis. In the current study, mRNA fragment species from BNT162b2 mRNA were isolated and characterized. The translational viability of intact and fragmented mRNA species was further explored using orthogonal expression systems to understand the risk of truncated spike protein or off-target antigen translation. The study demonstrates that mRNA fragments are primarily derived from premature transcriptional termination during manufacturing, and only full-length mRNA transcripts are viable for expression of the SARS-CoV-2 spike protein antigen.

The Race to Develop the Pfizer-BioNTech COVID-19 Vaccine: From the Pharmaceutical Scientists' Perspective.

At the outset of the coronavirus disease 2019 (COVID-19) pandemic, it was clear that a vaccine would be crucial for global health efforts. The Pfizer and BioNTech teams came together in a race against the virus, working to design, test, manufacture, and distribute a safe and efficacious vaccine in record time for people around the world. Here, we provide backstory commentary from the pharmaceutical scientist perspective on the challenges and solutions encountered in the development of the Pfizer-BioNTech mRNA COVID-19 vaccine (BNT162b2; b2; Comirnaty®; tozinameran). We discuss the foundational science that led to the decision to use an mRNA-based approach. We also describe key challenges in the identification of an optimal vaccine candidate and testing in clinical trials, the continuous efforts to improve the vaccine formulation in response to changing global health priorities and facilitate vaccine accessibility, and how vast quantities of vaccine doses were manufactured and safely delivered to every corner of the globe, all without compromising quality, science, and safety. The key to successfully delivering a safe and efficacious vaccine within nine months was a result of extraordinary, real-time, parallel effort and across-the-board collaboration between stakeholders on a global scale.

Strategies for Setting Patient-Centric Commercial Specifications for Biotherapeutic Products.

Commercial specifications for a new biotherapeutic product are a critical component of the product's overall control strategy that ensures safety and efficacy. This paper describes strategies for setting commercial specifications as proposed by a consortium of industry development scientists. The specifications for some attributes are guided by compendia and regulatory guidance. For other product quality attributes (PQAs), product knowledge and the understanding of attribute criticality built throughout product development should drive specification setting. The foundation of PQA knowledge is an understanding of potential patient impact through an assessment of potency, PK, immunogenicity and safety. In addition to PQA knowledge, the ability of the manufacturing process to consistently meet specifications, typically assessed through statistical analyses, is an important consideration in the specification-setting process. Setting acceptance criteria that are unnecessarily narrow can impact the ability to supply product or prohibit consideration of future convenient dosage forms. Patient-centric specifications enable appropriate control over higher risk PQAs to ensure product quality for the patient, and flexibility for lower risk PQAs for a sustainable supply chain. This paper captures common strategic approaches for setting specifications for standard biotherapeutic products such as monoclonal antibodies and includes considerations for ensuring specifications are patient centric.

Rational optimization of a monoclonal antibody for simultaneous improvements in its solution properties and biological activity.

Developability considerations should be integrated with lead engineering of antibody drug candidates in interest of their cost effective translations into medicines. To explore feasibility of this imperative, we have performed rational mutagenesis studies on a monoclonal antibody (MAB1) whose development was discontinued owing to manufacturability hurdles. Seven computationally designed variants of MAB1 containing single point (V44K, E59S, E59T and E59Y) and double (V44KE59S, V44KE59T and V44KE59Y) mutations in its light chain were produced in Chinese Hamster Ovary (CHO) cells and purified by using platform processes employed during commercial scale production of monoclonal antibodies. MAB1 and its variants were formulated in the same platform buffer and subjected to a battery of experiments to assess their solution behaviors, and biological activities. Five of the seven (71%) variants of MAB1 demonstrated improved biophysical attributes in multiple experimental testings. Contrary to the commonly expressed reservations about potential biological activity loss upon developability optimizations, the improvements in solution behavior of MAB1 also increased its biological activity up to ~180%. In particular, concentrate-ability and apparent solubility of V44KE59S improved to ~150% and ~160%, respectively. Its diffusion interaction parameter (kD) reduced to 28% and viscosity at ~100 mg/ml decreased to less than half of the corresponding values for MAB1. V44KE59S is also slightly more active and its transfections in CHO cells were more productive. It also degraded slower than MAB1 in three month long 25°C and 40°C formulation stability studies. These results open doors to an exciting realm of structure-based biologic drug design where developability and biological activity can be simultaneously optimized at the molecular engineering stages.

Establishing in vitro in vivo correlations to screen monoclonal antibodies for physicochemical properties related to favorable human pharmacokinetics.

Implementation of in vitro assays that correlate with in vivo human pharmacokinetics (PK) would provide desirable preclinical tools for the early selection of therapeutic monoclonal antibody (mAb) candidates with minimal non-target-related PK risk. Use of these tools minimizes the likelihood that mAbs with unfavorable PK would be advanced into costly preclinical and clinical development. In total, 42 mAbs varying in isotype and soluble versus membrane targets were tested in in vitro and in vivo studies. MAb physicochemical properties were assessed by measuring non-specific interactions (DNA- and insulin-binding ELISA), self-association (affinity-capture self-interaction nanoparticle spectroscopy) and binding to matrix-immobilized human FcRn (surface plasmon resonance and column chromatography). The range of scores obtained from each in vitro assay trended well with in vivo clearance (CL) using both human FcRn transgenic (Tg32) mouse allometrically projected human CL and observed human CL, where mAbs with high in vitro scores resulted in rapid CL in vivo. Establishing a threshold value for mAb CL in human of 0.32 mL/hr/kg enabled refinement of thresholds for each in vitro assay parameter, and using a combinatorial triage approach enabled the successful differentiation of mAbs at high risk for rapid CL (unfavorable PK) from those with low risk (favorable PK), which allowed mAbs requiring further characterization to be identified. Correlating in vitro parameters with in vivo human CL resulted in a set of in vitro tools for use in early testing that would enable selection of mAbs with the greatest likelihood of success in the clinic, allowing costly late-stage failures related to an inadequate exposure profile, toxicity or lack of efficacy to be avoided.

Characterization of FcγRIIIA effector cells used in in vitro ADCC bioassay: Comparison of primary NK cells with engineered NK-92 and Jurkat T cells.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action (MOA) of several therapeutic antibody drugs and evaluation in ADCC bioassays is important in antibody drug development and maintenance. Three types of effector cells now routinely used in bioassay evaluation of ADCC are natural killer cells from human donors (FcγRIIIA+primary NK), FcγRIIIA engineered NK-92 cells and FcγRIIIA/NFAT-RE/luc2 engineered Jurkat T cells. Engineered effector cells were developed to address need for improved precision and accuracy of classic NK cell ADCC bioassays. The main purpose of our study was to rationalize which of these ADCC effector cells best simulate the expected response in human subjects and to identify which effector cells and assays best fit ADCC bioassay needs during antibody drug development. We characterized differences between the effector cells and compared ADCC biological activities using the well-known humanized IgG1 antibody drug, trastuzumab. The three effector cell types studied expressed either V-158 or F-158 allotype of FcγRIIIA, hence six cell preparations were compared. Our results demonstrate highest surface expression of FcγRIIIA in primary NK and engineered NK-92 (V-158) cells with nearly all expressed on the cell surface. In contrast, expression in engineered Jurkat T cells was low with only a small percentage expressed on the cell surface. Studies evaluating binding of trastuzumab to effector cells demonstrated the highest affinity of FcγRIIIA in primary NK and NK-92 (V-158) cells. ADCC cytotoxicity studies showed greatest trastuzumab potency in primary NK and engineered NK-92 (V-158) cells and negligible cell lysis obtained using engineered Jurkat T cells. In contrast, the engineered Jurkat T (V-158) cells responded as effectively as primary NK (V/V) cells to nuclear factor of activated T cells (NFAT2) activation upon binding of trastuzumab to FcγRIIIA, demonstrating similar ADCC pathway activation in these cells despite the low surface expression of FcγRIIIA and its low affinity for trastuzumab. Dose-response range of trastuzumab in activation of NFAT2 (measured as pNFAT2 dephosphorylation) was very similar to response in classic ADCC assay for primary and NK-92 cells and to response in ADCC reporter assay for Jurkat T effector cells, bridging the assays. Trastuzumab potency in ADCC reporter assay using the engineered Jurkat T cells was close to that seen using either primary NK or engineered NK-92 cells in classic ADCC assay. In summary, all three effector cell systems differentially express FcγRIIIA and provide dose-dependent ADCC pathway activity, yet only primary NK and engineered NK-92 cells are capable of inducing ADCC-mediated cell lysis. Engineered Jurkat T effector cells have value to assure antibody manufacturing consistency and in other applications where accuracy and precision are important. For functional assessment of ADCC activity, primary NK or NK-92 (V-158) cells better reflect the physiologically relevant ADCC mechanism of action. As an engineered cell line, NK-92 cells may behave more reproducibly than primary NK, but this must be balanced with the objective for biological relevance in decisions on which NK cells to use in assay.