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.

Simultaneous Evaluation of a Vaccine Component Microheterogeneity and Conformational Integrity Using Native Mass Spectrometry and Limited Charge Reduction.

Analytical characterization of extensively modified proteins (such as haptenated carrier proteins in synthetic vaccines) remains a challenging task due to the high degree of structural heterogeneity. Native mass spectrometry (MS) combined with limited charge reduction allows these obstacles to be overcome and enables meaningful characterization of a heavily haptenated carrier protein CRM197 (inactivated diphtheria toxin conjugated with nicotine), a major component of a smoking cessation vaccine. The extensive conjugation results in a near-continuum distribution of ionic signal in electrospray ionization (ESI) mass spectra of haptenated CRM197 even after size-exclusion chromatographic fractionation. However, supplementing the ESI MS measurements with limited charge reduction of ionic populations selected within narrow m/z windows gives rise to well-resolved charge ladders, from which both masses and charge states of the ionic species can be readily deduced. Application of this technique to a research-grade material of CRM197/H7 conjugate not only reveals its marginal conformational stability (manifested by the appearance of high charge-density ions in ESI MS) but also establishes a role of the extent of haptenation as a major factor driving the loss of the higher order structure integrity. The unique information provided by native MS used in combination with limited charge reduction provides a strong argument for this technique to become a standard/required tool in the analytical arsenal in the field of biotechnology and biopharmaceutical analysis, where protein conjugates are becoming increasingly common.

Evolution of a comprehensive, orthogonal approach to sequence variant analysis for biotherapeutics.

Amino acid sequence variation in protein therapeutics requires close monitoring during cell line and cell culture process development. A cross-functional team of Pfizer colleagues from the Analytical and Bioprocess Development departments worked closely together for over 6 years to formulate and communicate a practical, reliable sequence variant (SV) testing strategy with state-of-the-art techniques that did not necessitate more resources or lengthen project timelines. The final Pfizer SV screening strategy relies on next-generation sequencing (NGS) and amino acid analysis (AAA) as frontline techniques to identify mammalian cell clones with genetic mutations and recognize cell culture process media/feed conditions that induce misincorporations, respectively. Mass spectrometry (MS)-based techniques had previously been used to monitor secreted therapeutic products for SVs, but we found NGS and AAA to be equally informative, faster, less cumbersome screening approaches. MS resources could then be used for other purposes, such as the in-depth characterization of product quality in the final stages of commercial-ready cell line and culture process development. Once an industry-wide challenge, sequence variation is now routinely monitored and controlled at Pfizer (and other biopharmaceutical companies) through increased awareness, dedicated cross-line efforts, smart comprehensive strategies, and advances in instrumentation/software, resulting in even higher product quality standards for biopharmaceutical products.

Practical approaches for overcoming challenges in heightened characterization of antibody-drug conjugates with new methodologies and ultrahigh-resolution mass spectrometry.

Antibody-drug conjugation strategies are continuously evolving as researchers work to improve the safety and efficacy of the molecules. However, as a part of process and product development, confirmation of the resulting innovative structures requires new, specialized mass spectrometry (MS) approaches and methods, as compared to those already established for antibody-drug conjugates (ADCs) and the heightened characterization practices used for monoclonal antibodies (mAbs), in order to accurately elucidate the resulting conjugate forms, which can sometimes have labile chemical bonds and more extreme chemical properties like hydrophobic patches. Here, we discuss practical approaches for characterization of ADCs using new methodologies and ultrahigh-resolution MS, and provide specific examples of these approaches. Denaturing conditions of typical liquid chromatography (LC)/MS analyses impede the successful detection of intact, 4-chain ADCs generated via cysteine site-directed chemistry approaches where hinge region disulfide bonds are partially reduced. However, this class of ADCs is detected intact reliably under non-denaturing size-exclusion chromatography/MS conditions, also referred to as native MS. For ADCs with acid labile linkers such as one used for conjugation of calicheamicin, careful selection of mobile phase composition is critical to the retention of intact linker-payload during LC/MS analysis. Increasing the pH of the mobile phase prevented cleavage of a labile bond in the linker moiety, and resulted in retention of the intact linker-payload. In-source fragmentation also was observed with typical electrospray ionization (ESI) source parameters during intact ADC mass analysis for a particular surface-accessible linker-payload moiety conjugated to the heavy chain C-terminal tag, LLQGA (via transglutaminase chemistry). Optimization of additional ESI source parameters such as cone voltages, gas pressures and ion transfer parameters led to minimal fragmentation and optimal sensitivity. Ultrahigh-resolution (UHR) MS, combined with reversed phase-ultrahigh performance (RP-UHP)LC and use of the FabRICATOR® enzyme, provides a highly resolving, antibody subunit-domain mapping method that allows rapid confirmation of integrity and the extent of conjugation. For some ADCs, the hydrophobic nature of the linker-payload hinders chromatographic separation of the modified subunit/domains or causes very late elution/poor recovery. As an alternative to the traditionally used C4 UHPLC column chemistry, a diphenyl column resulted in the complete recovery of modified subunit/domains. For ADCs based on maleimide chemistry, control of pH during proteolytic digestion is critical to minimize ring-opening. The optimum pH to balance digestion efficiency and one that does not cause ring opening needed to be established for successful peptide mapping.

Characterization of Apolipoprotein C3 (Apo C3) LNA/DNA Impurities and Degradation Products by LC-MS/MS.

Apolipoprotein C3 (Apo C3) LNA/DNA gapmer was evaluated under various stress and formulation conditions for the purpose of its development as a potential biotherapeutic for low density lipoprotein (LDL) lowering. Using ion-pairing (IP) reversed-phase (RP) liquid chromatography ultra-high resolution (UHR) tandem mass spectrometry (IP-RPLC-MS/MS), a combination of accurate mass measurements and collision-induced dissociation enabled in-depth characterization of Apo C3 LNA/DNA oligonucleotide, in particular the inherent impurities following synthesis and degradation products after exposure to stress conditions. In this study, oligonucleotide samples were stressed under different pH and UV exposure conditions. The primary impurities in Apo C3 LNA/DNA were losses of nucleotide moieties from both the 5'- and 3'-terminus leading to n-1, n-2, etc. species. Desulfurization and depurination were observed in Apo C3 LNA/DNA after a week under UV light stress conditions at low pH. Guanine oxidation and dimerization were the primary degradation products detected under UV light exposure for 1 week at high pH. The effect of antioxidants on the levels of these degradation products was evaluated under neutral pH conditions. In the presence of all antioxidants, levels of guanine oxidation and desulfurization under tested conditions were the same as those in the unstressed sample, except for sodium ascorbate. The thorough understanding of the Apo C3 LNA/DNA oligonucleotide structure, its impurities, and degradation products laid the foundation for the successful formulation development of this novel biotherapeutic modality.

The Dual Role of Lipids of the Lipoproteins in Trumenba, a Self-Adjuvanting Vaccine Against Meningococcal Meningitis B Disease.

Trumenba (bivalent rLP2086) is a vaccine licensed for the prevention of meningococcal meningitis disease caused by Neisseria meningitidis serogroup B (NmB) in individuals 10-25 years of age in the USA. The vaccine is composed of two factor H binding protein (fHbp) variants that were recombinantly expressed in Escherichia coli as native lipoproteins: rLP2086-A05 and rLP2086-B01. The vaccine was shown to induce potent bactericidal antibodies against a broad range of NmB isolates expressing fHbp that were different in sequence from the fHbp vaccine antigens. Here, we describe the characterization of the vaccine antigens including the elucidation of their structure which is characterized by two distinct motifs, the polypeptide domain and the N-terminal lipid moiety. In the vaccine formulation, the lipoproteins self-associate to form micelles driven by the hydrophobicity of the lipids and limited by the size of the folded polypeptides. The micelles help to increase the structural stability of the lipoproteins in the absence of bacterial cell walls. Analysis of the lipoproteins in Toll-like receptor (TLR) activation assays revealed their TLR2 agonist activity. This activity was lost with removal of the O-linked fatty acids, similar to removal of all lipids, demonstrating that this moiety plays an adjuvant role in immune activation. The thorough understanding of the structure and function of each moiety of the lipoproteins, as well as their relationship, lays the foundation for identifying critical parameters to guide vaccine development and manufacture.

On-line coupling of size exclusion chromatography with mixed-mode liquid chromatography for comprehensive profiling of biopharmaceutical drug product.

A methodology based on on-line coupling of size exclusion chromatography (SEC) with mixed-mode liquid chromatography (LC) has been developed. The method allows for simultaneous measurement of a wide range of components in biopharmaceutical drug products. These components include the active pharmaceutical ingredient (protein) and various kinds of excipients such as cations, anions, nonionic hydrophobic surfactant and hydrophilic sugars. Dual short SEC columns are used to separate small molecule excipients from large protein molecules. The separated protein is quantified using a UV detector at 280 nm. The isolated excipients are switched, online, to the Trinity P1 mixed-mode column for separation, and detected by an evaporative light scattering detector (ELSD). Using a stationary phase with 1.7 µm particles in SEC allows for the use of volatile buffers for both SEC and mix-mode separation. This facilitates the detection of different excipients by ELSD and provides potential for online characterization of the protein with mass spectrometry (MS). The method has been applied to quantitate protein and excipients in different biopharmaceutical drug products including monoclonal antibodies (mAb), antibody drug conjugates (ADC) and vaccines.