A High Threshold of Biotherapeutic Aggregate Numbers is Needed to Induce an Immunogenic Response In Vitro, In Vivo, and in the Clinic.

BACKGROUND AND PURPOSE: There is concern that subvisible aggregates in biotherapeutic drug products pose a risk to patient safety. We investigated the threshold of biotherapeutic aggregates needed to induce immunogenic responses. METHODS AND RESULTS: Highly aggregated samples were tested in cell-based assays and induced cellular responses in a manner that depended on the number of particles. The threshold of immune activation varied by disease state (cancer, rheumatoid arthritis, allergy), concomitant therapies, and particle number. Compared to healthy donors, disease state patients showed an equal or lower response at the late phase (7 days), suggesting they may not have a higher risk of responding to aggregates. Xeno-het mice were used to assess the threshold of immune activation in vivo. Although highly aggregated samples (~ 1,600,000 particles/mL) induced a weak and transient immunogenic response in mice, a 100-fold dilution of this sample (~ 16,000 particles/mL) did not induce immunogenicity. To confirm this result, subvisible particles (up to ~ 18,000 particles/mL, containing aggregates and silicone oil droplets) produced under representative administration practices (created upon infusion of a drug product through an IV catheter) did not induce a response in cell-based assays or appear to increase the rate of adverse events or immunogenicity during phase 3 clinical trials. CONCLUSION: The ability of biotherapeutic aggregates to elicit an immune response in vitro, in vivo, and in the clinic depends on high numbers of particles. This suggests that there is a high threshold for aggregates to induce an immunogenic response which is well beyond that seen in standard biotherapeutic drug products.

Observation of Heavy-Chain C-Terminal Des-GK Truncation in Recombinant and Human Endogenous IgG4.

Therapeutic IgG mAbs have shown presence of three variations of their heavy chain C-termini, including the unprocessed C-terminal lysine, the processed C-terminal lysine, and C-terminal amidation. These variants are also present in endogenous human IgGs, although the level of unprocessed C-terminal lysine is very low. Here we report a new heavy-chain C-terminal variant, i.e., the des-GK truncation, which exists in both recombinant and endogenous human IgG4. The des-GK truncation was found in negligible amount in IgG1, IgG2 and IgG3 subclasses. Observation of a significant level of heavy-chain C-terminal des-GK truncation in endogenous human IgG4 suggests that low level of this variant present in therapeutic IgG4 is unlikely to be a safety concern.

Process development of a SARS-CoV-2 nanoparticle vaccine.

One of the outcomes from the global COVID-19 pandemic caused by SARS-CoV-2 has been an acceleration of development timelines to provide treatments in a timely manner. For example, it has recently been demonstrated that the development of monoclonal antibody therapeutics from vector construction to IND submission can be achieved in five to six months rather than the traditional ten-to-twelve-month timeline using CHO cells [1], [2]. This timeline is predicated on leveraging existing, robust platforms for upstream and downstream processes, analytical methods, and formulation. These platforms also reduce; the requirement for ancillary studies such as cell line stability, or long-term product stability studies. Timeline duration was further reduced by employing a transient cell line for early material supply and using a stable cell pool to manufacture toxicology study materials. The development of non-antibody biologics utilizing traditional biomanufacturing processes in CHO cells within a similar timeline presents additional challenges, such as the lack of platform processes and additional analytical assay development. In this manuscript, we describe the rapid development of a robust and reproducible process for a two-component self-assembling protein nanoparticle vaccine for SARS-CoV-2. Our work has demonstrated a successful academia-industry partnership model that responded to the COVID-19 global pandemic quickly and efficiently and could improve our preparedness for future pandemic threats.

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.

Observation of Heavy-Chain C-Terminal Amidation in Human Endogenous IgG.

Therapeutic IgG mAbs expressed from Chinese hamster ovary (CHO) cells are known to contain three C-terminal variants in their heavy chains, namely, the unprocessed C-terminal lysine, the processed C-terminal lysine, and C-terminal amidation. Although the presence of C-terminal amidation in CHO-expressed IgGs is well studied, the biological impact of the variant on the safety and efficacy of biotherapeutics has not been well understood. To further our biological understanding of C-terminal amidation, we analyzed a series of IgG samples, including both endogenous human IgGs as well as recombinant IgGs of different subclasses expressed from both CHO and murine cell lines, for their heavy-chain C-terminal variants by LC-MS/MS based peptide mapping. The results demonstrate that heavy-chain C-terminal amidation is a common variant occurring in IgG of all four subclasses (IgG1, IgG2, IgG3 and IgG4). The variant is generally present in recombinant IgG mAbs expressed from CHO cell lines but not in IgG mAbs expressed from murine cell lines, whereas the IgGs expressed from murine cell lines contain a much larger amount of unprocessed C-terminal lysine. Additionally, a significant amount of heavy-chain C-terminal amidation is observed in endogenous human IgGs, indicating that small amount of the variant present in therapeutic IgGs does not pose a safety concern.

A Mass Spectrometric Characterization of Light-Induced Modifications in Therapeutic Proteins.

During the development of a therapeutic protein, its quality attributes that pertain to the primary structure must be appropriately characterized, commonly by LC-MS/MS peptide mapping experiments. Extracting attribute information from LC-MS/MS data requires knowledge of the attribute of interest. Therefore, it is important to understand all potential modifications on the therapeutic proteins. In this work, we performed UV and visible light irradiation experiments on several therapeutic proteins, with or without the presence of a photosensitizer. Light-induced modifications were detected and characterized by tryptic digestion followed by LC-MS/MS analysis. A list of potential light-induced modifications, with their respective mass changes, was obtained. These modifications are primarily on methionine, tryptophan, histidine, cysteine, tyrosine and phenylalanine residues. Many of these modifications have not been previously reported on therapeutic proteins. Our findings therefore provide a database of potential light-induced modifications that would enable the routine characterization of light-induced modifications on therapeutic proteins.

Analytical Quality by Design, Life Cycle Management, and Method Control.

Analytical methods are utilized throughout the biopharmaceutical and vaccines industries to conduct research and development, and to help control manufacturing inputs and outputs. These analytical methods should continuously provide quality data to support decisions while managing the remaining of risk and uncertainty. Analytical quality by design (AQbD) can provide a systematic framework to achieve a continuously validated, robust assay as well as life cycle management. AQbD is rooted in ICH guidelines Q8 and Q9 that were translated to the analytical space through several white papers as well as upcoming USP 1220 and ICH Q14. In this white paper, we expand on the previously published concepts of AQbD by providing additional context for implementation in relation to ICH Q14. Using illustrative examples, we describe the AQbD workflow, its relation to traditional approaches, and potential pathways for ongoing, real-time verification. We will also discuss challenges with respect to implementation and regulatory strategies.

Observation and Mitigation of Leachables from Non-Product Contact Materials in Electromechanical Delivery Devices for Biotechnology Products.

Battery-powered drug delivery devices are widely used as primary containers for storing and delivering therapeutic protein products to improve patient compliance and quality of life. Compared to conventional delivery approaches such as pre-filled syringes, battery-powered devices are more complex in design requiring new materials/components for proper functionality, which could cause potential product safety and quality concerns from the extractable and leachables (E&L) of the new materials/components. In this study, E&L assessments were performed on a battery-powered delivery device during the development and qualification of the device, where novel compound 2­hydroxy-2-methylpropiophenone (HMPP) and related compounds were observed in both E&L. The source of the HMPP and related compounds was identified to be the nonproduct contact device batteries, in which HMPP photo-initiator was used as a curing agent in the battery sealant to prevent leakage of the battery electrolytes. Toxicology assessment was performed, which showed the levels of HMPP observed in the device lots were acceptable relative to the permitted daily exposure. A drug product HMPP spike study was also performed, where no product impact was observed. Based on these assessments, an action threshold and specification limits could be established as a control strategy, if needed, to mitigate the potential risks associate with the observed leachables. As a full resolution, seven battery candidates from different suppliers were screened and one new battery was successfully qualified for the delivery devices. Overall, the holistic E&L approach was fully successful in the development and qualification of the battery-powered devices for biotherapeutic products delivery ensuring product quality and patient safety. Non-product contact materials are commonly rated as low or no risk and typically considered as out of scope of E&L activities for delivery systems following industry benchmark and regulatory agency guidance. This case study is novel as it brings into attention the materials that might not normally be in consideration during the development process. It is highly recommended to understand materials in the context of intended use on a case-by-case basis and not to generalize to ensure successful development and qualification.

Development and Validation of a HPLC-UV Method for Urea and Related Impurities.

Urea is used in biopharmaceutical manufacturing processes for the purification of therapeutic proteins, for cleaning columns, and for refolding proteins after purification. The urea used for such purposes is typically USP grade material obtained from commercial sources and further characterization is required prior to use, such as determination of purity and identity. For this purpose, a robust analytical method is needed that can characterize the known organic impurities of urea. However, the existing methods show high assay variability and are not able to resolve all known organic impurities as desired for accurate quantification. In the present manuscript we developed a new high-performance liquid chromatography method with UV detection for the separation of urea and its impurities (biuret, cyanuric acid, and triuret). The method performance characteristics evaluated for urea and biuret were specificity, linearity, accuracy, identity, precision, and robustness and the newly developed method met all predefined performance acceptance criteria.

Direct mass spectrometric characterization of disulfide linkages.

Disulfide linkage is critical to protein folding and structural stability. The location of disulfide linkages for antibodies is routinely discovered by comparing the chromatograms of the reduced and non-reduced peptide mapping with location identification confirmed by collision-induced dissociation (CID) mass spectrometry (MS)/MS. However, CID product spectra of disulfide-linked peptides can be difficult to interpret, and provide limited information on the backbone region within the disulfide loop. Here, we applied an electron-transfer dissociation (ETD)/CID combined fragmentation method that identifies the disulfide linkage without intensive LC comparison, and yet maps the disulfide location accurately. The native protein samples were digested using trypsin for proteolysis. The method uses RapiGest SF Surfactant and obviates the need for reduction/alkylation and extensive sample manipulation. An aliquot of the digest was loaded onto a C4 analytical column. Peptides were gradient-eluted and analyzed using a Thermo Scientific LTQ Orbitrap Elite mass spectrometer for the ETD-triggered CID MS 3 experiment. Survey MS scans were followed by data-dependent scans consisting of ETD MS2 scans on the most intense ion in the survey scan, followed by 5 MS3 CID scans on the 5 most intense ions in the ETD MS2 scan. We were able to identify the disulfide-mediated structural variants A and A/B forms and their corresponding disulfide linkages in an immunoglobulin G2 monoclonal antibody with λ light chain (IgG2λ), where the location of cysteine linkages were unambiguously determined.

Investigation of the free heavy chain homodimers of a monoclonal antibody.

Monoclonal antibodies (mAbs) are composed of two heavy chain (HC) and two light chain (LC) polypeptides. The proper folding and assembly of HC and LC is critical for antibody production. Current dogma indicates that the free HCs are retained in the endoplasmic reticulum (ER) unless assembled with LCs into antibodies, while the LCs on the other hand can be secreted as free monomer or dimer molecules. In this study, high levels of extracellular HC homodimers (7%-45%) were observed in the cell culture media during cell line development for mAb1. Excellent correlation (R2  > 0.9) between the level of free HC homodimers and the percentage of high molecular weight species indicates that the free HC homodimers might be causative of unwanted aggregation. Due to the different surface charge of HC homodimer and fully assembled antibodies, the unwanted extracellular HC homodimers were successfully removed by downstream processing, through a cation exchange chromatography step. Reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS) analysis of the cell culture media from different MTX-amplified pools indicated that insufficient expression of LC is one potential root cause for the high level of free HC homodimers. The level of free HC homodimers decreased significantly (3%-25%) after retransfecting the MTX amplified pools with additional LC gene. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:738-745, 2018.

A View on the Importance of "Multi-Attribute Method" for Measuring Purity of Biopharmaceuticals and Improving Overall Control Strategy.

Today, we are experiencing unprecedented growth and innovation within the pharmaceutical industry. Established protein therapeutic modalities, such as recombinant human proteins, monoclonal antibodies (mAbs), and fusion proteins, are being used to treat previously unmet medical needs. Novel therapies such as bispecific T cell engagers (BiTEs), chimeric antigen T cell receptors (CARTs), siRNA, and gene therapies are paving the path towards increasingly personalized medicine. This advancement of new indications and therapeutic modalities is paralleled by development of new analytical technologies and methods that provide enhanced information content in a more efficient manner. Recently, a liquid chromatography-mass spectrometry (LC-MS) multi-attribute method (MAM) has been developed and designed for improved simultaneous detection, identification, quantitation, and quality control (monitoring) of molecular attributes (Rogers et al. MAbs 7(5):881-90, 2015). Based on peptide mapping principles, this powerful tool represents a true advancement in testing methodology that can be utilized not only during product characterization, formulation development, stability testing, and development of the manufacturing process, but also as a platform quality control method in dispositioning clinical materials for both innovative biotherapeutics and biosimilars.

Chemical and Biophysical Characteristics of Monoclonal Antibody Solutions Containing Aggregates Formed during Metal Catalyzed Oxidation.

PURPOSE: To physicochemically characterize and compare monoclonal antibody (mAb) solutions containing aggregates generated via metal catalyzed oxidation (MCO). METHODS: Two monoclonal IgG2s (mAb1 and mAb2) and one monoclonal IgG1 (rituximab) were exposed to MCO with the copper/ascorbic acid oxidative system, by using several different methods. The products obtained were characterized by complementary techniques for aggregate and particle analysis (from oligomers to micron sized species), and mass spectrometry methods to determine the residual copper content and chemical modifications of the proteins. RESULTS: The particle size distribution and the morphology of the protein aggregates generated were similar for all mAbs, independent of the MCO method used. There were differences in both residual copper content and in chemical modification of specific residues, which appear to be dependent on both the protein sequence and the protocol used. All products showed a significant increase in the levels of oxidized His, Trp, and Met residues, with differences in extent of modification and specific amino acid residues modified. CONCLUSION: The extent of total oxidation and the amino acid residues with the greatest oxidation rate depend on a combination of the MCO method used and the protein sequence.

Improved sequence variant analysis strategy by automated false positive removal.

Sequence variant analysis (SVA) is critical in therapeutic protein development because it ensures the absence of genetic mutations of a production clone or high-level misincorporations during cell culture. While software for searching sequence variants from mass spectrometry data are available, effectively distinguishing true positives from a large number of false positives in the reported hits or identifications found in the error tolerant search mode is a challenge. This verification process must be done manually and can take several days or even weeks to accomplish. We report here the use of a Perl-based script to evaluate every identified hit to remove the false positives from the search results of PepFinder™ (also known as MassAnalyzer) based on orthogonal criteria. Our data show that the false positives from PepFinder™ output were reduced ∼4-fold without loss of accuracy in the detection of true identifications, representing a more than 70% reduction in time compared with the manual data verification process.

Engineering an IgG Scaffold Lacking Effector Function with Optimized Developability.

IgG isotypes can differentially bind to Fcγ receptors and complement, making the selection of which isotype to pursue for development of a particular therapeutic antibody important in determining the safety and efficacy of the drug. IgG2 and IgG4 isotypes have significantly lower binding affinity to Fcγ receptors. Recent evidence suggests that the IgG2 isotype is not completely devoid of effector function, whereas the IgG4 isotype can undergo in vivo Fab arm exchange leading to bispecific antibody and off-target effects. Here an attempt was made to engineer an IgG1-based scaffold lacking effector function but with stability equivalent to that of the parent IgG1. Care was taken to ensure that both stability and lack of effector function was achieved with a minimum number of mutations. Among the Asn297 mutants that result in lack of glycosylation and thus loss of effector function, we demonstrate that the N297G variant has better stability and developability compared with the N297Q or N297A variants. To further improve the stability of N297G, we introduced a novel engineered disulfide bond at a solvent inaccessible location in the CH2 domain. The resulting scaffold has stability greater than or equivalent to that of the parental IgG1 scaffold. Extensive biophysical analyses and pharmacokinetic (PK) studies in mouse, rat, and monkey further confirmed the developability of this unique scaffold, and suggest that it could be used for all Fc containing therapeutics (e.g. antibodies, bispecific antibodies, and Fc fusions) requiring lack of effector function or elimination of binding to Fcγ receptors.

Structural Characterization of a Monoclonal Antibody-Maytansinoid Immunoconjugate.

Structural characterization was performed on an antibody-drug conjugate (ADC), composed of an IgG1 monoclonal antibody (mAb), mertansine drug (DM1), and a noncleavable linker. The DM1 molecules were conjugated through nonspecific modification of the mAb at solvent-exposed lysine residues. Due to the nature of the lysine conjugation process, the ADC molecules are heterogeneous, containing a range of species that differ with respect to the number of DM1 per antibody molecule. The DM1 distribution profile of the ADC was characterized by electrospray ionization mass spectrometry (ESI-MS) and capillary isoelectric focusing (cIEF), which showed that 0-8 DM1s were conjugated to an antibody molecule. By taking advantage of the high-quality MS/MS spectra and the accurate mass detection of diagnostic DM1 fragment ions generated from the higher-energy collisional dissociation (HCD) approach, we were able to identify 76 conjugation sites in the ADC, which covered approximately 83% of all the putative conjugation sites. The diagnostic DM1 fragment ions discovered in this study can be readily used for the characterization of other ADCs with maytansinoid derivatives as payload. Differential scanning calorimetric (DSC) analysis of the ADC indicated that the conjugation of DM1 destabilized the C(H)2 domain of the molecule, which is likely due to conjugation of DM1 on lysine residues in the C(H)2 domain. As a result, methionine at position 258 of the heavy chain, which is located in the C(H)2 domain of the antibody, is more susceptible to oxidation in thermally stressed ADC samples when compared to that of the naked antibody.

Capillary zone electrophoresis method for a highly glycosylated and sialylated recombinant protein: development, characterization and application for process development.

A purity method based on capillary zone electrophoresis (CZE) has been developed for the separation of isoforms of a highly glycosylated protein. The separation was found to be driven by the number of sialic acids attached to each isoform. The method has been characterized using orthogonal assays and shown to have excellent specificity, precision and accuracy. We have demonstrated the CZE method is a useful in-process assay to support cell culture and purification development of this glycoprotein. Compared to isoelectric focusing (IEF), the CZE method provides more quantitative results and higher sample throughput with excellent accuracy, qualities that are required for process development. In addition, the CZE method has been applied in the stability testing of purified glycoprotein samples.

Development of a human antibody tolerant mouse model to assess the immunogenicity risk due to aggregated biotherapeutics.

We describe a novel human immunoglobulin G2 (IgG2 )-tolerant and immune-competent heterozygous mouse model (Xeno-het) developed by crossbreeding a human Ig-tolerized XenoMouse® with a C57BL/6J wild-type mouse. The Xeno-het mouse expresses both mouse and human immunoglobulin G (IgG) genes, resulting in B-cells expressing human and mouse IgG, and secretion of human and mouse Ig into serum. This model was utilized to evaluate the immunogenicity risk of aggregated and chemically modified human antibodies. The mice were tested for their ability to break tolerance to self-tolerant monomeric antibodies. Aggregates made by mechanical stirring elicited an anti-drug antibody (ADA) response, but did not induce a robust and long-term memory B and T-cell response. Chemically modified antibodies made by oxidation were only weak and transient inducers of an immune response, as measured by a lack of both an ADA response and a B-cell antigen-specific response. Aggregate size was an important characteristic, as specific-sized protein-coated beads were able to elicit an immune response. We propose the use of this model to identify risk factors such as aggregation during manufacturing at early development for an increased potential immunogenicity risk.

Assessment of stable isotope incorporation into recombinant proteins.

Stable isotope labeling combined with mass spectrometry has been widely used in a diverse set of applications in the biochemistry and biomedical fields. When stable isotope-labeled proteins are produced via metabolic labeling of cell culture, a comprehensive assessment of the labeling pattern is imperative. In this study, we present a set of mass spectrometry-based bioanalytical tools developed for quantitatively tracing the levels of the stable isotopes incorporated into the recombinant proteins (monoclonal antibodies and Fc fusion proteins expressed in different host systems) that include total mass analysis, peptide mapping analysis, and amino acid analysis. We show that these three mass spectrometry-based analytical methods have distinctive advantages and limitations and that they are mutually complementary in evaluating the quality of stable isotope-labeled proteins. In addition, we show that the analytical techniques developed here are powerful tools to provide valuable insights into studying cell metabolism and performing flux analysis during cell culture.