Impact of Polysorbate 80 Grade on the Interfacial Properties and Interfacial Stress Induced Subvisible Particle Formation in Monoclonal Antibodies.

Polysorbate 80 is a nonionic surfactant that is added to therapeutic protein formulations to mitigate protein particle formation when subjected to various mechanical stresses. Variations in the PS80 grade has recently sparked questions surrounding the effect of oleic acid content (OAC) on surfactant's ability to mitigate interface-induced protein particle formation when stressed. In this work, a Langmuir trough was used to apply interfacial dilatational stress to two IgG molecules (mAb1 and mAb2) in formulations containing Chinese pharmacopeia (CP) and multicompendial (MC) grades of PS80.

Investigation of anomalous charge variant profile reveals discrete pH-dependent conformations and conformation-dependent charge states within the CDR3 loop of a therapeutic mAb.

During the development of a therapeutic monoclonal antibody (mAb-1), the charge variant profile obtained by pH-gradient cation exchange chromatography (CEX) contained two main peaks, each of which exhibited a unique intrinsic fluorescence profile and demonstrated inter-convertibility upon reinjection of isolated peak fractions. Domain analysis of mAb-1 by CEX and liquid chromatography-mass spectrometry indicated that the antigen-binding fragment chromatographed as two separate peaks that had identical mass. Surface plasmon resonance binding analysis to antigen demonstrated comparable kinetics/affinity between these fractionated peaks and unfractionated starting material.

Hybrid mode of hydrophobic interaction chromatography of monoclonal antibodies and related biomolecules: Influence of elution conditions on chromatographic performance using poly (alkyl aspartamide) silica columns.

A hybrid mode of hydrophobic interaction chromatography (HHIC) is an emerging analytical technique for the separation of biomolecules under non-denaturing conditions that combines elements of conventional hydrophobic interaction and reversed-phase chromatography. This article explores the impact of mobile phase composition such as salt concentration and organic modifier on the separation of therapeutic monoclonal antibodies and related large biomolecules using poly (alkyl aspartamide) silica HIC columns. The initial mobile phase salt concentration had a significant impact on the separation of a mixture of large biomolecules demonstrating that the relationship of elution and salt concentration was more complex than in conventional HIC.

Cyclization of N-Terminal Glutamic Acid to pyro-Glutamic Acid Impacts Monoclonal Antibody Charge Heterogeneity Despite Its Appearance as a Neutral Transformation.

Pyroglutamic acid (pyroGlu) is commonly observed at the N-terminus of therapeutic monoclonal antibodies. Notably, the term "pyroGlu" refers to a single product that could originate from the cyclization of either an N-terminal glutamine or an N-terminal glutamic acid. This is an important and easily overlooked distinction that has major implications on the charge variant nature of a pyroGlu relative to its uncyclized form.

Comparison of imaged capillary isoelectric focusing and cation exchange chromatography for monitoring dextrose-mediated glycation of monoclonal antibodies in infusion solutions.

Intravenous (IV) infusion of therapeutic proteins typically involves dilution of the formulated product into infusion media such as normal saline or dextrose, 5% m/v in water. We report results from a rigorous evaluation of imaged capillary isoelectric focusing (iCIEF) for monitoring dextrose-mediated glycation of proteins in IV infusion solutions. In addition to detecting stable Amadori glycation products, iCIEF was able to detect the labile Schiff base (SB) glycation adducts since the equilibrium with free dextrose is maintained on capillary.

Pulse Proteolysis: An Orthogonal Tool for Protein Formulation Screening.

Protein formulation stability is difficult to predict a priori and generally involves long-term stability studies. It is of interest to develop an analytical method that can predict stability trends reliably. Here, pulse proteolysis was evaluated as an analytical tool to predict solution-state stability in different formulations.

Improving Mass Spectral Quality of Monoclonal Antibody Middle-Up LC-MS Analysis by Shifting the Protein Charge State Distribution.

Monoclonal antibodies (mAbs) are experiencing accelerated development in the pharmaceutical industry. Utilization of middle-up LC-MS methodology can provide detailed characterization of mAbs via reduction and/or enzymatic cleavage of the mAb into smaller protein fragments. However, under typical LC-MS conditions, these fragments, especially the more heterogeneous heavy chain, can present charge state distributions (CSD) featuring a severe interference in the low mass-to-charge (m/z) region in the mass spectrum, adversely impacting spectral quality of these proteins and ultimately the deconvoluted mass spectrum.

Enablement of the direct analysis of excipients in monoclonal antibody formulations through the incorporation of a wide pore C18 protein trap with hydrophilic interaction liquid chromatography.

Establishing and maintaining the correct formulation composition is essential for ensuring the stability of biopharmaceutical drug products. A barrier to the routine assessment of excipient concentration is the lack of convenient and robust methods for the direct analysis of solutions containing high protein concentrations. To address this need an HPLC method was developed utilizing a wide-pore C18 guard column to trap proteins in-line with a hydrophilic interaction liquid chromatographic column to separate excipients.

Identification of leachables observed in the size exclusion chromatograms of a low concentration product stored in prefilled syringes.

Requisite leachables testing of pharmaceutical products is commonly conducted with pre-defined analytical methods on a subset of materials intended to be representative of the marketed product. Throughout product development, leachables may occasionally be detected in other methods not specifically intended for monitoring such impurities. We have identified two leachables, ethyl 4-ethoxybenzoate (E4E) and 2,6-di(t-butyl)-4-hydroxy-4-methyl-2,5-cyclohexadien-1-one (BHT-OH) in a low concentration product stored in prefilled syringes (PFS).

Drug-to-antibody determination for an antibody-drug-conjugate utilizing cathepsin B digestion coupled with reversed-phase high-pressure liquid chromatography analysis.

Antibody drug conjugates or ADCs are currently being evaluated for their effectiveness as targeted chemotherapeutic agents across the pharmaceutical industry. Due to the complexity arising from the choice of antibody, drug and linker; analytical methods for release and stability testing are required to provide a detailed understanding of both the antibody and the drug during manufacturing and storage. The ADC analyzed in this work consists of a tubulysin drug analogue that is randomly conjugated to lysine residues in a human IgG1 antibody.

Exploitation of the size-exclusion effect of reversed-phase high performance liquid chromatography for the direct analysis of diethylene triamine pentaacetic acid in therapeutic monoclonal antibody formulations.

Monoclonal antibodies (mAb) are being widely studied for the treatment of cancers and other diseases. The mAb is typically in a solution formulation and administered as an intravenous infusion. Ready-to-use solutions are favored for their clinical convenience but they can potentially suffer from a shorter shelf life due to accelerated rates of some forms of degradation such as oxidation, relative to lyophilized formulations.

Structural characterization of low level degradants in aztreonam injection and an innovative approach to aid HPLC method validation.

Three new degradants have been identified from drug product and active pharmaceutical ingredient stability samples of aztreonam, a marketed synthetic monocyclic beta-lactam antibiotic. The degradants were detected following the implementation of a new, more selective HPLC method for the determination of impurities and degradants. The new method was developed in response to changes in the regulatory requirement for mature products.

Trace level liquid chromatography tandem mass spectrometry quantification of the mutagenic impurity 2-hydroxypyridine N-oxide as its dansyl derivative.

A derivatization LC-MS/MS method was developed and qualified for the trace level quantification of 2-hydroxypyridine N-oxide (HOPO). HOPO is a coupling reagent used in the syntheses of active pharmaceutical ingredients (APIs) to form amide bonds. HOPO was recently confirmed to generate a positive response in a GLP Ames bacterial-reverse-mutation test, classifying it as a mutagenic impurity and as such requiring its control in APIs to the threshold of toxicological concern (TTC).

A novel route to recognizing quaternary ammonium cations using electrospray mass spectrometry.

Characterizing and elucidating structures is a commonplace and necessary activity in the pharmaceutical industry with mass spectrometry and NMR being the primary tools for analysis. Although many functional groups are readily identifiable, quaternary ammonium cations have proven to be difficult to unequivocally identify using these techniques. Due to the lack of an N-H bond, quaternary ammonium groups can only be detected in the (1)H NMR spectra by weak signals generated from long-range (14)N-H coupling, which by themselves are inconclusive evidence of a quaternary ammonium functional group.

Improving the efficiency of quantitative (1)H NMR: an innovative external standard-internal reference approach.

The classical internal standard quantitative NMR (qNMR) method determines the purity of an analyte by the determination of a solution containing the analyte and a standard. Therefore, the standard must meet the requirements of chemical compatibility and lack of resonance interference with the analyte as well as a known purity. The identification of such a standard can be time consuming and must be repeated for each analyte.

Determination of the molecular weight of poly(ethylene glycol) in biological samples by reversed-phase LC-MS with in-source fragmentation.

PEGylation has been widely used to improve the biopharmaceutical properties of therapeutic proteins and peptides. Previous studies have used multiple analytical techniques to determine the fate of both the therapeutic molecule and unconjugated poly(ethylene glycol) (PEG) after drug administration. A straightforward strategy utilizing liquid chromatography-mass spectrometry (LC-MS) to characterize high-molecular weight PEG in biologic matrices without a need for complex sample preparation is presented.

Mitigation of signal suppression caused by the use of trifluoroacetic acid in liquid chromatography mobile phases during liquid chromatography/mass spectrometry analysis via post-column addition of ammonium hydroxide.

A method has been developed to reduce the mass spectrometric ion signal suppression associated with the use of TFA as an additive in LC mobile phases. Through post-column infusion of diluted NH(4)OH solution to LC eluents, the ammonium ion introduced causes the neutral analyte-TFA ion pair to dissociate which consequently releases the protonated analyte as free ions into the gas phase (through regular electrospray ionization mechanisms). An ion signal improvement from 1.

Separation and detection of bis-maleimide-polyethylene glycol and mono-maleimide-polyethylene glycol by reversed-phase high pressure liquid chromatography.

Monofunctional maleimide polyethylene glycol (mono-mal-PEG) with average molecular weight up to 40 kDa can be used as a raw material for the PEGylation of therapeutic proteins. A possible impurity in this raw material which needs to be controlled is the bisfunctional maleimide-PEG, which has a similar average molecular weight to mono-mal-PEG. Chromatographic separation and detection of low level bis-mal-PEG in mono-mal-PEG presents a major challenge because of the polydispersity of the analytes and the minor difference between the desired mono-mal-PEG and the bis-mal-PEG impurity.

Development of a two-step tier-2 dissolution method for blinded overencapsulated erlotinib tablets using UV fiber optic detection.

Measuring dissolution of a comparator drug overencapsulated in a hard gelatin shell is necessary when determining performance of the native and blinded formulations. However, the gelatin in the shell may form cross-links upon storage at stressed conditions, resulting in slow dissolution of the encapsulated drug. The aim of this study was to develop a dissolution approach for a hard-gelatin overencapsulated formulation of a comparator drug, erlotinib, which can overcome cross linking of the capsule shell.

A combined desorption ionization by charge exchange (DICE) and desorption electrospray ionization (DESI) source for mass spectrometry.

A source that couples the desorption ionization by charge exchange (DICE) and desorption electrospray ionization (DESI) techniques together was demonstrated to broaden the range of compounds that can be analyzed in a single mass spectrometric experiment under ambient conditions. A tee union was used to mix the spray reagents into a partially immiscible blend before this mixture was passed through a conventional electrospray (ES) probe capillary. Using this technique, compounds that are ionized more efficiently by the DICE method and those that are ionized better with the DESI procedure could be analyzed simultaneously.

Effect of mobile phase pH, aqueous-organic ratio, and buffer concentration on electrospray ionization tandem mass spectrometric fragmentation patterns: implications in liquid chromatography/tandem mass spectrometric bioanalysis.

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) based on selected reaction monitoring (SRM) is the standard methodology in quantitative analysis of administered xenobiotics in biological samples. Utilizing two SRM channels during positive electrospray ionization (ESI) LC/MS/MS method development for a drug compound containing two basic functional groups, we found that the response ratio (SRM1/SRM2) obtained using an acidic mobile phase was dramatically different from that obtained using a basic mobile phase. This observation is different from the well-established phenomenon of mobile phase affecting the [M+H](+) response, which is directly related to the amount of the [M+H](+) ions produced during the ionization.

Evading metal adduct formation during desorption-ionization mass spectrometry.

An investigation into the propensity of metal adduct formation in the recently developed Desorption Ionization by Charge Exchange (DICE) mass spectrometric technique has demonstrated that this method could be utilized to minimize spectral complications caused by metal adducts. For example, peaks for sodium and other metal adducts were not observed in the mass spectra acquired by the ambient-pressure DICE technique from samples deposited on a solid surface, even after the salt content of samples was deliberately increased. A mass spectrum recorded from a urine sample by this technique showed peaks only for the proton adducts of urea and creatinine.

Reactivity of gaseous sodiated ions derived from benzene dicarboxylate salts toward residual water in the collision gas.

The sodium adduct of disodium salts of benzene dicarboxylic acids (m/z 233), when subjected to collision-induced dissociation (CID), undergoes a facile loss of CO(2) to produce an ion of m/z 189, which retains all the three sodium atoms of the precursor. The CID spectrum of this unusual m/z 189 ion shows significant peaks at m/z 167, 63 and 85. The enigmatic m/z 167 ion, which appeared to represent a loss of a 22-Da neutral fragment from the precursor ion is in fact a fragment produced by the interaction of the m/z 189 ion with traces of water present in the collision gas.

Desorption ionization by charge exchange (DICE) for sample analysis under ambient conditions by mass spectrometry.

An ambient pressure ionization technique for mass spectrometric analysis of substances present on solid surfaces was developed. A nebulized spray containing molecular ions of a solvent such as toluene can be generated by passing the solvent through a stainless steel capillary held at a high voltage. When the stream of charged droplets produced in this way is directed onto a solid surface, the analytes present on the surface are desorbed and ionized by a charge exchange process.

Generation of gas-phase sodiated arenes such as [(Na3(C6H4)+] from benzene dicarboxylate salts.

Upon collision-induced activation, gaseous sodium adducts generated by electrospray ionization of disodium salts of 1,2- 1,3-, and 1,4-benzene dicarboxylic acids (m/z 233) undergo an unprecedented expulsion of CO(2) by a rearrangement process to produce an ion of m/z 189 in which all three sodium atoms are retained. When isolated in a collision cell of a tandem-in-space mass spectrometer, and subjected to collision-induced dissociation (CID), only the m/z 189 ions derived from the meta and para isomers underwent a further CO(2) loss to produce a peak at m/z 145 for a sodiated arene of formula (Na(3)C(6)H(4))(+). This previously unreported m/z 145 ion, which is useful to differentiate meta and para benzene dicarboxylates from their ortho isomer, is in fact the sodium adduct of phenelenedisodium.