A Spike-Control Approach that Evaluates High Resolution Mass Spectrometry-Based Sequence Variant Analytical Method Performance for Therapeutic Proteins.

An amino acid sequence variant (SV) is defined as an unintended amino acid substitution in protein drug products. SVs contribute to product heterogeneity and can potentially impact product quality, safety, immunogenicity, and efficacy. The analysis of biotherapeutics for SVs is important throughout the product life cycle including clone selection, development of nutrient feed strategies, commercial manufacturing process, and post-approval changes to monitor product quality. The proposed analytical procedure for SVs consists of both qualitative (identification of SVs) and quantitative (quantitation of identified SVs) components. The complexities of SV analysis and the variety of current procedures highlight the need for a systematic approach for assessing the capability of these methodologies to reliably identify and quantitate SVs in biotherapeutics. We described here a "spike-control" approach for evaluating SV analytical procedure. The concept was adopted from quality control samples routinely used in analytical procedure validation. One FDA approved monoclonal antibody (mAb) was spiked with accurate amounts of highly homologous mAb to create mAb samples containing low yet accurate levels of "artificial" SVs. Spike-control samples were denatured, reduced, alkylated, digested and then analyzed by high resolution Orbitrap mass spectrometry. In silico analysis revealed four single amino acid differences between the two mAbs that could be used to represent SVs in the spike-control samples. All four "artificial" SVs were reliably identified by the current workflow. Analytical range (0.01% to 2%), accuracy and precision of identified SVs have also been evaluated. Overall, spike-control sample(s) helped to demonstrate that the SV analytical procedure (i.e., sample preparation, LC separation, mass spectrometry determinations and bioinformatic software) was fit for purpose and suitable for the identification and quantitation of SVs at a pre-determined threshold.

Ion/ion Charge Inversion/Attachment in Conjunction with Dipolar DC Collisional Activation as a Selective Screen for Sulfo- and Phosphopeptides.

We describe a gas-phase approach for the rapid screening of polypeptide anions for phosphorylation or sulfonation based on binding strengths to guanidinium-containing reagent ions. The approach relies on the generation of a complex via reaction of mixtures of deprotonated polypeptide anions with dicationic guanidinium-containing reagent ions and subsequent dipolar DC collisional activation of the complexes. The relative strengths of the electrostatic interactions of guanidinium with deprotonated acidic sites follows the order carboxylate

Characterization of Homopolymer Distributions via Direct Infusion ESI-MS/MS using Wide Mass-to-Charge Windows and Gas-Phase Ion/Ion Reactions.

A hallmark of electrospray ionization (ESI) of large polymeric molecules is its tendency to generate charge state distributions. When a distribution of polymers is subjected to ESI, the charge state distribution of each component can lead to a mass spectrum composed of a highly congested mixture of ions with overlapping mass-to-charge (m/z) ratios. When the polymers are composed of a common monomeric unit (i.e., a homopolymer), the overlap of the charge state distributions of the polymer components can give rise to striking spectral patterns with a dense central cluster of peaks having similar m/z values and wing-like patterns on either side. We refer to the central cluster of peaks as an "Emerald City," with a nod to the Wizard of Oz, combining the wings as an "Emerald City pattern". The Emerald City pattern can appear in the mass spectrum of any homopolymer with distributions of charge states and sizes. Various parameters were studied individually for their contributions to the appearance of Emerald City patterns. Dextran samples were used to demonstrate the spectral pattern experimentally, and a web-based tool was developed to validate the findings. We also proposed to use direct infusion ESI-MS coupled with segmented m/z windows that encompass Emerald Cities followed by gas-phase proton transfer reactions for characterizing poly disperse synthetic polymer samples. Poly(ethylenimine) samples were used as model systems to demonstrate the approach. The proposed strategy improves sample characterization relative to conventional zero-charge deconvolution or proton transfer reactions without prior mass-selected m/z windows.

Effect of formulation and peptide folding on the fibrillar aggregation, gelation, and oxidation of a therapeutic peptide.

The physical and chemical stability of therapeutic peptides presents challenges in developing robust formulations. The stability of the formulation affects product safety, efficacy and quality. Therefore, an understanding of the effects of formulation variables on the peptide's conformational structure and on its possible physical and chemical degradation is vital. To this end, computational and experimental analysis were employed to investigate the impact of formulation, peptide folding and product handling on oxidation, fibrillar aggregation and gelation of teriparatide. Teriparatide was used as a model drug due to the correlation of its conformation in solution with its pharmacological activity. Fibrillar aggregation and gelation were monitored using four orthogonal techniques. An innovative, automated platform coupled with ion mobility mass spectrometry was used for profiling chemical degradants. Increases in teriparatide concentration, pH, and ionic strength were found to increase the rate of fibrillar aggregation and gelation. Conversely, an increase in peptide folding and stabilization of the folded structures was found to decrease the rate of fibrillar aggregation and gelation. Moreover, the rate of oxidation was found to be inversely related to its solution concentration and extent of peptide folding. The present study provides an insight into formulation strategies designed to reduce the potential risk of physical and chemical degradation of peptides with a defined conformation.

Generation of Multiply Charged Protein Anions from Multiply Charged Protein Cations via Gas-Phase Ion/Ion Reactions.

We report a novel charge inversion ion/ion reaction that converts multiply charged protein cations to multiply charged protein anions via a single ion/ion collision using highly charged anions derived from nanoelectrospray ionization (nESI) of hyaluronic acids (HAs). This type of charge inversion reaction is demonstrated with cations derived from cytochrome c, apo-myoglobin, and carbonic anhydrase (CA) cations. For example, the reaction has been demonstrated to convert the [CA+22H]22+ carbonic anhydrase cation to a distribution of anions as high in absolute charge as [CA-19H]19-. Ion/ion reactions involving multiply charged ions of opposite polarity have previously been observed to result predominantly in the attachment of the reactant ions. All mechanisms for ion/ion charge inversion involving low energy ions proceed via the formation of a long-lived complex. Factors that underlie the charge inversion of protein cations to high anionic charge states in reaction with HA anions are hypothesized to include: (i) the relatively high charge densities of the HA anions that facilitate the extraction of multiple protons from the protein leading to multiply charged protein anions, (ii) the relatively high sum of absolute charges of the reactants that leads to high initial energies in the ion/ion complex, and (iii) the relatively high charge of the ion/ion complex following the multiple proton transfers that tends to destabilize the complex.