Investigation of a failed DNA assay leads to identification of an unexpected contaminant in a commonly used chromatography buffer.

For mammalian cell-derived recombinant biotherapeutics, controlling host cell DNA levels below a threshold is a regulatory requirement to ensure patient safety. DNA removal during drug substance manufacture is accomplished by a series of chromatography-based purification steps and a qPCR-based analytical method is most used to measure DNA content in the purified drug substance to enable material disposition. While the qPCR approach is mature and its application to DNA measurement is widespread in the industry, it is susceptible to trace levels of process-related contaminants that are carried forward. In this study, we observed failures in spike recovery studies that are an integral component of the qPCR-based DNA testing, suggesting the presence of an inhibitory compound in the sample matrix. We generated hypotheses around the origin of the inhibitory compound and generated multiple sample matrices and deployed a suite of analytical techniques including Raman and NMR spectroscopy to determine the origin and identity of the inhibitory compound. The caustic wash step and depth filter extractables were ruled out as root causes after extensive experimentation and DNA testing. Subsequently, 2-(N-morpholino)ethanesulfonic acid (MES), a buffer used in the chromatography unit operations, was identified as the source of the contaminant. A 500-fold concentration followed by Raman and NMR spectroscopy analysis revealed the identity of the inhibitory compound as polyvinyl sulfone (PVS), an impurity that originates in the MES manufacturing process. We have implemented PVS concentration controls for incoming MES raw material, and our work highlights the need for rigor in raw material qualification and control.

State-of-the-art and emerging trends in analytical approaches to pharmaceutical-product commercialization.

The biopharmaceutical landscape continues to evolve rapidly, and associated modality complexity and the need to improve molecular understanding require concomitant advances in analytical approaches used to characterize and release the product. The Product Quality Attribute Assessment (PQAA) and Quality Target Product Profile (QTPP) frameworks help catalog and translate molecular understanding to process and product-design targets, thereby enabling reliable manufacturing of high-quality product. The analytical target profile forms the basis of identifying best-fit analytical methods for attribute measurement and continues to be successfully used to develop robust analytical methods for detailed product characterization as well as release and stability testing. Despite maturity across multiple testing platforms, advances continue to be made, several with the potential to alter testing paradigms. There is an increasing role for mass spectrometry beyond product characterization and into routine release testing as seen by the progress in multi-attribute methods and technologies, applications to aggregate measurement, the development of capillary zone electrophoresis (CZE) coupled with mass spectrometry (MS) and capillary isoelectric focusing (CIEF) with MS for measurement of glycans and charged species, respectively, and increased application to host cell protein measurement. Multitarget engaging multispecific modalities will drive advances in bioassay platforms and recent advances both in 1- and 2-D NMR approaches could make it the method of choice for characterizing higher-order structures. Additionally, rigorous understanding of raw material and container attributes is necessary to complement product understanding, and these collectively can enable robust supply of high-quality product to patients.

Observation and Mitigation of Lamellar Silica Particles Formed in Pharmaceutical Products Packaged in Glass Vials.

A new type of lamellae-like particles was observed in protein based liquid therapeutic protein drug product (DP) packaged in standard (STD) and delamination controlled (DC) Type IB glass vials stored at 2-8°C as early as two weeks after manufacture. These particles were determined to be remarkably different from lamellae in not only in their chemical composition, but in the mechanism by which these are formed. The lamellae-like particles were an ultra-thin (< 200 nm) film, appeared curled, sheet-like, folded with no defined edges identified as lamellar silica composed of silica and polysorbate 80 (PS 80). It was also observed that the lamellar silica particles, when formed in a given drug product lot, not only were observed in a small percentage of vials, but also remained at low (≤ 5) numbers in affected vials, often decreasing in number over time. This is in contrast to the large number of commonly reported glass lamellae (hundreds per vial) observed in vials prone to delamination with a glass vial interior showing a delaminated inner surface. In this case study, evidence from low Si leachable levels in solution and various surface analytical techniques supported the conclusion that there was neither delamination nor early signs of glass delamination like reaction zones occurring in those impacted vials, regardless. A mechanism for particle formation was hypothesized and experimentally confirmed. Lamellar silica particles are composed of an admixture of condensed silica and PS 80 deposited on the interior walls of glass vials, which form and may be released into solution over time. The root cause was determined to be conditions present during preparation of the vials for drug product filling, specifically the vial washing and depyrogenation steps. These conditions are known to make glass vials prone to delamination; in this case study, they resulted in interactions between the glass and PS 80 present in the formulation. Incomplete drying of the glass vials during depyrogenation in closed ovens was confirmed as the contributing factors that led to lamellar silica particle formation via the studies of silicate spiked into the DC Type IB glass vials filled with the mAb DP in which lamellar silica particles were observed. Prevention of lamellar silica particles formation was successfully achieved through optimization of the duration and pressure of air blow during the vial washing and drying process in a depyrogenation oven. This was evidenced by the lack of appearance of the lamellar silica particles over 48 months for the DP lots filled post optimization. Additionally, the formation of lamellar silica was also mitigated by changing the vial washing process from a closed oven process to a tunnel process, which allowed for improved air flow and hence better drying of the vial primary container.

Non-targeted characterization of attributes affecting antibody-FcγRIIIa V158 (CD16a) binding via online affinity chromatography-mass spectrometry.

Antibodies facilitate targeted cell killing by engaging with immune cells such as natural killer cells through weak binding interactions with Fcγ receptors on the cell surface. Here, we evaluate the binding affinity of the receptor FcγRIIIa V158 (CD16a) for several therapeutic antibody classes, isoforms, and Fc-fusion proteins using an immobilized receptor affinity liquid chromatography (LC) approach coupled with online mass spectrometry (MS) detection. Aglycosylated FcγRIIIa was used in the affinity chromatography and compared with published affinities using glycosylated receptors. Affinity LC-MS differentiated the IgG1 antibodies primarily according to their Fc glycosylation patterns, with highly galactosylated species having greater affinity for the immobilized receptors and thus eluting later from the column (M5< G0F < G0 afucosylated ≅ G1F < G2F). Sialylated species bound weaker to their asialylated counterparts as reported previously. High mannose glycoforms bound weaker than G0F, contrary to previously published studies using glycosylated receptors. Also, increased receptor binding affinity associated with afucosylated antibodies was not observed with the aglycosylated FcγRIIIa. This apparent difference from previous findings highlighted the importance of the glycans on the receptors for mediating stronger binding interactions. Characterization of temperature-stressed samples by LC-MS peptide mapping revealed over 200 chemical and post-translational modifications, but only the Fc glycans, deamidation of EU N325, and an unknown modification to either proline or cysteine residues of the hinge region were found to have a statistically significant impact on binding.Abbreviations: Antibody-dependent cell-mediated cytotoxicity (ADCC), chimeric antigen receptor (CAR), Chinese hamster ovary (CHO), dithiothreitol (DTT), electrospray ionization (ESI), hydrogen-deuterium exchange (HDX), filter aided-sample preparation (FASP), Fcγ receptor (FcγR), fragment crystallizable (Fc), high-pressure liquid chromatography (HPLC), immunoglobulin G (IgG), liquid chromatography (LC), monoclonal antibody (mAb), mass spectrometry (MS), natural killer (NK), N-glycolylneuraminic acid (NGNA), N-acetylneuraminic acid (NANA), principal component analysis (PCA), surface plasmon resonance (SPR), trifluoroacetic acid (TFA), and extracted mass chromatogram (XMC).

Enabling development, manufacturing, and regulatory approval of biotherapeutics through advances in mass spectrometry.

Rapid technological advances have significantly improved the capability, versatility, and robustness of mass spectrometers which has led to them playing a central role in the development, characterization, and regulatory filings of biopharmaceuticals. Their application spans the entire continuum of drug development, starting with discovery research through product development, characterization, and marketing authorization and continues well into product life cycle management. The scope of application extends beyond traditional protein characterization and includes elements like clone selection, cell culture physiology and bioprocess optimization, investigation support, and process analytical technology. More recently, advances in the MS-based multi-attribute method are enabling the introduction of MS in a cGMP environment for routine release and stability testing. While most applications of MS to date have been for monoclonal antibodies, the successes and learnings should translate to the characterization of next-gen biotherapeutics where modalities like multispecifics could be more prevalent. In this review, we describe the most significant advances in MS and correlate them to the broad spectrum of applications to biotherapeutic development. We anticipate rapid technological improvements to continue that will further accelerate widespread deployment of MS, thereby elevating our overall understanding of product quality and enabling attribute-focused product development.

Identification of critical chemical modifications and paratope mapping by size exclusion chromatography of stressed antibody-target complexes.

Therapeutic proteins including antibodies and Fc-fusion proteins undergo a large number of chemical modifications during cell culture, purification, storage and in human circulation. They are also exposed to harsh conditions during stress studies, including elevated temperature, extremes of pH, forced oxidation, physiological pH, UV light to assess the possible degradation pathways and suitability of methods for detecting them. Some of these modifications are located on residues in binding regions, leading to loss of binding and potency and classified as critical quality attributes. Currently, criticality of modifications is assessed by a laborious process of collecting antibody fractions from the soft chromatography techniques ion exchange and hydrophobic interaction chromatography and characterizing the fractions one-by-one for potency and chemical modifications. Here, we describe a method for large-scale, parallel identification of all critical chemical modifications in one experiment. In the first step, the antibody is stressed by one or several stress methods. It is then mixed with target protein and separated by size-exclusion chromatography (SEC) on bound antibody-target complex and unbound antibody. Peptide mapping of fractions and statistical analysis are performed to identify modifications on amino acid residues that affect binding. To identify the modifications leading to slight decreases in binding, competitive SEC of antibody and antigen mixtures was developed and described in a companion study by Shi et al, where target protein is provided at lower level, below the stoichiometry. The newly described method was successfully correlated to crystallography for assessing criticality of chemical modifications and paratope mapping. It is more sensitive to low-level modifications, better streamlined and platform ready.

Applicability of total reflection X-ray fluorescence (TXRF) as a screening platform for pharmaceutical inorganic impurity analysis.

Palladium (Pd) is extensively used in pharmaceutical small molecule drug substance processes, however it must be removed prior to release of the active pharmaceutical ingredient (API). Evaluation of four TXRF instruments and configurations were compared to ICP-MS instrumentation for trace metal analysis, most importantly for Pd. Standards and six pharmaceutical drug substances, triprolidine HCl, diphenhydramine HCl, chlorpheniramine maleate, pseudoephedrine HCl, ephedrine sulfate, and scopolamine HBr, were analyzed to determine linearity, sensitivity, accuracy, and precision for Pd plus Cr, Fe, Cu, Rh, and Pt versus interferences, particularly from Cl, S, and Ar, on the various X-ray fluorescence lines. Irrespective of instrument platform, in general X-ray sources capable of accessing Pd-K lines were found to be most effective in determination of Pd in APIs.

A novel approach to determine water content in DMSO for a compound collection repository.

The quality of a corporate compound collection can be significantly affected by a complex combination of storage and operational processing factors. Water content in DMSO solutions is one factor that is of great interest as it can affect solubility, degradation, and freeze-thaw cycle parameters. To the authors' knowledge, this is the first report of using near-infrared (NIR) spectroscopy to assess water content in DMSO compound stock solutions within the common storage vessel format of polypropylene microtubes. The precision and accuracy of the NIR technique was benchmarked against a Karl Fisher titration method, and a correlation coefficient was determined to be 0.985 over a range of 1% to 10% water in DMSO by weight. The advantages of the NIR technique include accuracy, precision, speed, nondestructiveness, and the capability of assessing compounds under in situ storage conditions within microtubes. In this report, the authors demonstrate the accuracy and precision of using NIR to assess water content in DMSO solutions and present a case study to demonstrate the utility of the technique to aid in assessing a pharmaceutical compound collection.