Optimizing PCSK9 Inhibitor Integration for Cardiovascular Disease Management in Pakistan.

Cardiovascular illnesses (CVDs), particularly Coronary Artery Disease (CAD) and Ischemic Heart Disease (IHD), are major global health burdens, with a growing incidence in Pakistan. The development of PCSK9 inhibitors offers encouraging advantages in lowering LDL cholesterol and lowering cardiovascular risk, even though conservative treatments are still essential. However, access to them is severely hampered by their high cost, especially in environments with little resources. The financial limitations and scarcity of healthcare resources while examining the difficulties in obtaining PCSK9 inhibitors in Pakistan is essential. In order to develop solutions for affordability and fair access, it emphasizes the urgent need for multi-stakeholder collaboration, including governmental action, healthcare sector involvement, and pharmaceutical company engagement. It also emphasizes the need for data-specific research and the use of PCSK9 inhibitors in conventional treatment protocols.

Development of time-resolved fluoroimmunoassay with enhanced fluorescence reaction for the quantitation of atezolizumab in plasma.

Atezolizumab (ATZ) is a human monoclonal antibody, which has been granted multiple approvals from the US Food and Drug Administration (FDA) for the immunotherapy of different types of cancer. This study describes the prototype of a time-resolved fluoroimmunoassay (TRFIA) for the quantitation of ATZ in plasma. The assay involved the non-competitive binding of ATZ to its specific antigen [programmed death-ligand 1 (PD-L1) protein]. The immune complex formed on the inner surface of the assay plate wells was quantified by anti-human secondary antibody labeled with a chelate of europium-ethylenediaminetetraacetic acid. The enhanced fluorescence signal was generated by an enhanced fluorescence solution composed of thenoyltrifluoroacetone, trioctylphosphine oxide, and Triton X-100. The conditions of the TRFIA were refined, and its optimum procedures were established. The assay was validated in accordance with the immunoassay validation guidelines, and all the validation parameters were acceptable. The working range of the assay was 20-1000 pg mL-1, and its limit of quantitation was 20 pg mL-1. The assay was applied to the quantitation of ATZ in plasma samples with satisfactory accuracy and precision. The proposed TRFIA has significant benefits over the existing methodologies for the quantitation of ATZ in clinical settings.

Comparison of N-Glycopeptide to Released N-Glycan Abundances and the Influence of Glycopeptide Mass and Charge States on N-Linked Glycosylation of IgG Antibodies.

We report the comparison of mass-spectral-based abundances of tryptic glycopeptides to fluorescence abundances of released labeled glycans and the effects of mass and charge state and in-source fragmentation on glycopeptide abundances. The primary glycoforms derived from Rituximab, NISTmAb, Evolocumab, and Infliximab were high-mannose and biantennary complex galactosylated and fucosylated N-glycans. Except for Evolocumab, in-source ions derived from the loss of HexNAc or HexNAc-Hex sugars are prominent for other therapeutic IgGs. After excluding in-source fragmentation of glycopeptide ions from the results, a linear correlation was observed between fluorescently labeled N-glycan and glycopeptide abundances over a dynamic range of 500. Different charge states of human IgG-derived glycopeptides containing a wider variety of abundant attached glycans were also investigated to examine the effects of the charge state on ion abundances. These revealed a linear dependence of glycopeptide abundance on the mass of the glycan with higher charge states favoring higher-mass glycans. Findings indicate that the mass spectrometry-based bottom-up approach can provide results as accurate as those of glycan release studies while revealing the origin of each attached glycan. These site-specific relative abundances are conveniently displayed and compared using previously described glycopeptide abundance distribution spectra "GADS" representations. Mass spectrometry data are available from the MAssIVE repository (MSV000093562).

Unraveling the complex interplay between anti-tumor immune response and autoimmunity mediated by B cells and autoantibodies in the era of anti-checkpoint monoclonal antibody therapies.

The intricate relationship between anti-tumor immunity and autoimmunity is a complex yet crucial aspect of cancer biology. Tumor microenvironment often exhibits autoimmune features, a phenomenon that involves natural autoimmunity and the induction of humoral responses against self-antigens during tumorigenesis. This induction is facilitated by the orchestration of anti-tumor immunity, particularly within organized structures like tertiary lymphoid structures (TLS). Paradoxically, a significant number of cancer patients do not manifest autoimmune features during the course of their illness, with rare instances of paraneoplastic syndromes. This discrepancy can be attributed to various immune-mediated locks, including regulatory or suppressive immune cells, anergic autoreactive lymphocytes, or induction of effector cells exhaustion due to chronic stimulation. Overcoming these locks holds the risk to induce autoimmune mechanisms during cancer progression, a phenomenon notably observed with anti-immune checkpoint therapies, in contrast to more conventional treatments like chemotherapy or radiotherapy. Therefore, the challenge arises in managing immune-related adverse events (irAEs) induced by immune checkpoint inhibitors treatment, as decoupling them from the anti-tumor activity poses a significant clinical dilemma. This review summarizes recent advances in understanding the link between B-cell driven anti-tumor responses and autoimmune reactions in cancer patients, and discusses the clinical implications of this relationship.

Human iPSC-Based Model of COPD to Investigate Disease Mechanisms, Predict SARS-COV-2 Outcome, and Test Preventive Immunotherapy.

Chronic inflammation and dysregulated repair mechanisms after epithelial damage have been implicated in chronic obstructive pulmonary disease (COPD). However, the lack of ex vivo-models that accurately reflect multicellular lung tissue hinders our understanding of epithelial-mesenchymal interactions in COPD. Through a combination of transcriptomic and proteomic approaches applied to a sophisticated in vitro iPSC-alveolosphere with fibroblasts model, epithelial-mesenchymal crosstalk was explored in COPD and following SARS-CoV-2 infection. These experiments profiled dynamic changes at single-cell level of the SARS-CoV-2-infected alveolar niche that unveiled the complexity of aberrant inflammatory responses, mitochondrial dysfunction, and cell death in COPD, which provides deeper insights into the accentuated tissue damage/inflammation/remodeling observed in patients with SARS-CoV-2 infection. Importantly, this 3D system allowed for the evaluation of ACE2-neutralizing antibodies and confirmed the potency of this therapy to prevent SARS-CoV-2 infection in the alveolar niche. Thus, iPSC-alveolosphere cultured with fibroblasts provides a promising model to investigate disease-specific mechanisms and to develop novel therapeutics.

Recent Advancements in AAV-Vectored Immunoprophylaxis in the Nonhuman Primate Model.

Monoclonal antibodies (mAbs) are important treatment modalities for preventing and treating infectious diseases, especially for those lacking prophylactic vaccines or effective therapies. Recent advances in mAb gene cloning from naturally infected or immunized individuals has led to the development of highly potent human mAbs against a wide range of human and animal pathogens. While effective, the serum half-lives of mAbs are quite variable, with single administrations usually resulting in short-term protection, requiring repeated doses to maintain therapeutic concentrations for extended periods of time. Moreover, due to their limited time in circulation, mAb therapies are rarely given prophylactically; instead, they are generally administered therapeutically after the onset of symptoms, thus preventing mortality, but not morbidity. Adeno-associated virus (AAV) vectors have an established record of high-efficiency in vivo gene transfer in a variety of animal models and humans. When delivered to post-mitotic tissues such as skeletal muscle, brain, and heart, or to organs in which cells turn over slowly, such as the liver and lungs, AAV vector genomes assume the form of episomal concatemers that direct transgene expression, often for the lifetime of the cell. Based on these attributes, many research groups have explored AAV-vectored delivery of highly potent mAb genes as a strategy to enable long-term expression of therapeutic mAbs directly in vivo following intramuscular or intranasal administration. However, clinical trials in humans and studies in nonhuman primates (NHPs) indicate that while AAVs are a powerful and promising platform for vectored immunoprophylaxis (VIP), further optimization is needed to decrease anti-drug antibody (ADA) and anti-capsid antibody responses, ultimately leading to increased serum transgene expression levels and improved therapeutic efficacy. The following review will summarize the current landscape of AAV VIP in NHP models, with an emphasis on vector and transgene design as well as general delivery system optimization. In addition, major obstacles to AAV VIP, along with implications for clinical translation, will be discussed.

Mirikizumab for the treatment of moderate to severe ulcerative colitis.

Despite a growing number of available therapeutic options for ulcerative colitis (UC), up to 50% of patients do not respond to initial treatment or lose response over time, highlighting the need for novel therapies. The IL-23 pathway has emerged as an important therapeutic target for UC. Mirikizumab is a humanized IgG4 monoclonal antibody against the p19 subunit of IL-23, dosed intravenously during induction and subcutaneously during maintenance. It is effective for the induction and maintenance of remission in moderately to severely active UC, including patients with prior failure of biological or tofacitinib therapy. Like other IL-23 antagonists, mirikizumab has a favorable safety profile. It is the first agent of its class to receive regulatory approval for moderately to severely active UC in Europe.

Despite a growing number of available therapeutic options for ulcerative colitis (UC), up to 50% of patients do not respond to initial treatment or lose response over time, highlighting the need for novel therapies. A molecule promoting inflammation in the colon called IL-23 is a promising target for new drugs that treat UC. Mirikizumab is an antibody that works against a portion of IL-23 and thus suppresses inflammation in the colon. Mirikizumab was shown to be effective in alleviating symptoms and resolving inflammation of the colon in patients with UC. The drug was safe and well tolerated by patients. Mirikizumab is the first drug of its kind to receive approval for UC in Europe.

Development of a highly sensitive chemiluminescence immunoassay using a novel signal-enhanced detection system for quantitation of durvalumab, an immune-checkpoint inhibitor monoclonal antibody used for immunotherapy of lung cancer.

Durvalumab (DUR) is a human monoclonal antibody used for the immunotherapy of lung cancer. It is a novel immune-checkpoint inhibitor, which blocks the programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) proteins and works to promote the normal immune responses that attack the tumour cells. To support the pharmacokinetic (PK) studies, therapeutic drug monitoring (TDM) and refining the safety profile of DUR, an efficient assay is required, preferably immunoassay. This study describes, for the first time, the development of a highly sensitive chemiluminescence immunoassay (CLIA) for the quantitation of DUR in plasma samples with enhanced chemiluminescence detection system. The CLIA protocol was conducted in 96-microwell plates and involved the non-competitive binding reaction of DUR to its specific antigen (PD-L1 protein). The immune complex of DUR with PD-L1 formed onto the inner surface of the assay plate wells was quantified by a chemiluminescence (CL)-producing horseradish peroxidase (HRP) reaction. The reaction employed 4-(1,2,4-triazol-1-yl)phenol (TRP) as an efficient enhancer of the HRP-luminol-hydrogen peroxide (H2O2) CL reaction. The optimum protocol of the proposed CLIA was established, and its validation parameters were assessed as per the guidelines for the validation of immunoassays for bioanalysis. The working dynamic range of the assay was 10-800 pg mL-1 with a limit of detection (LOD) of 10.3 pg mL-1. The assay enables the accurate and precise quantitation of DUR in human plasma at a concentration as low as 30.8 pg mL-1. The CLIA protocol is simple and convenient; an analyst can analyse several hundreds of samples per working day. This high throughput property enables the processing of many samples in clinical settings. The proposed CLIA has a significant benefit in the quantitation of DUR in clinical settings for assessment of its PK, TDM and refining the safety profile.

Diagnostic Flow Cytometry in the Era of Targeted Therapies: Lessons from Therapeutic Monoclonal Antibodies and Chimeric Antigen Receptor T-cell Adoptive Immunotherapy.

Therapeutic monoclonal antibodies (therapeutic mAb) and adoptive immunotherapy have become increasingly more common in the treatment of hematolymphoid neoplasms, with practical implications for diagnostic flow cytometry. Their use can reduce the sensitivity of flow cytometry for populations of interest owing to downregulation/loss of the target antigen, competition for the target antigen, or lineage switch. Expanded flow panels, marker redundancy, and exhaustive gating strategies can overcome this limitation. Therapeutic mAb have been reported to cause pseudo-light chain restriction, and awareness of this potential artifact is key. Established guidelines do not yet exist for antigen expression by flow cytometry for therapeutic purposes.

NKG2A Immune Checkpoint in Vδ2 T Cells: Emerging Application in Cancer Immunotherapy.

Immune regulation has revolutionized cancer treatment with the introduction of T-cell-targeted immune checkpoint inhibitors (ICIs). This successful immunotherapy has led to a more complete view of cancer that now considers not only the cancer cells to be targeted and destroyed but also the immune environment of the cancer cells. Current challenges associated with the enhancement of ICI effects are increasing the fraction of responding patients through personalized combinations of multiple ICIs and overcoming acquired resistance. This requires a complete overview of the anti-tumor immune response, which depends on a complex interplay between innate and adaptive immune cells with the tumor microenvironment. The NKG2A was revealed to be a key immune checkpoint for both Natural Killer (NK) cells and T cells. Monalizumab, a humanized anti-NKG2A antibody, enhances NK cell activity against various tumor cells and rescues CD8 αß T cell function in combination with PD-1/PD-L1 blockade. In this review, we discuss the potential for targeting NKG2A expressed on tumor-sensing human γδ T cells, mostly on the specific Vδ2 T cell subset, in order to emphasize its importance and potential in the development of new ICI-based therapeutic approaches.

Parathyroid hormone-related protein (PTHrP) and malignancy.

PTHrP (parathyroid hormone related protein) is an important mediator of malignancy-related tumor progression and hypercalcemia that shares considerable homology and functionality with parathyroid hormone. In this chapter, we review what has been elucidated to date regarding PTHrP's role in malignancies. Starting with a review of calcium metabolism and regulation, we then summarize the discovery and structure of PTHrP and development of sensitive immunoassays for specific measurement. Subsequently, we explore its role in tumor progression, with emphasis on the primary tumor as well as skeletal and non-osseus metastases. We then consider the clinical implications of PTHrP in cancer before concluding with a discussion of both established and potential treatments for malignancy associated hypercalcemia and bone metastases.

Ocrelizumab quantitation by liquid chromatography-tandem mass spectrometry.

Introduction: Ocrelizumab is a monoclonal anti-CD20 antibody approved for the treatment of multiple sclerosis (MS). The clinical value of therapeutic drug monitoring (TDM) for this antibody in treatment of MS is unknown, and an adequately specific and precise quantitation method for ocrelizumab in patient serum could facilitate investigation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based quantitation methods have been shown to have higher analytic specificity and precision than enzyme-linked immunosorbent assays. Objectives: To establish and validate an LC-MS/MS-based quantitation method for ocrelizumab. Methods: We present an LC-MS/MS-based quantitation method using immunocapture purification followed by trypsinization and analysis by a triple quadrupole mass analyzer obtaining results within the same day. Results: We found that the ocrelizumab peptide GLEWVGAIYPGNGDTSYNQK (Q1/Q3 Quantifier ion: 723.683+/590.77 y112+ Qualifier ion: 723.683+/672.30 y122+) can be used for quantitation and thereby developed a method for quantifying ocrelizumab in human serum with a quantitation range of 1.56 to 200 µg/mL. The method was validated in accordance with EMA requirements in terms of selectivity, carry-over, lower limit of quantitation, calibration curve, accuracy, precision and matrix effect. Ocrelizumab serum concentrations were measured in three MS patients treated with ocrelizumab, immediately before and after ocrelizumab infusion, with additional sampling after 2, 4, 8 and 12 weeks. Measured serum concentrations of ocrelizumab showed expected values for both Cmax and drug half-life over the sampled time period. Conclusion: We have established a reliable quantitation method for serum ocrelizumab that can be applied in clinical studies, facilitating the evaluation of ocrelizumab TDM in MS.

3D Models as a Tool to Assess the Anti-Tumor Efficacy of Therapeutic Antibodies: Advantages and Limitations.

Therapeutic monoclonal antibodies (mAbs) are an emerging and very active frontier in clinical oncology, with hundred molecules currently in use or being tested. These treatments have already revolutionized clinical outcomes in both solid and hematological malignancies. However, identifying patients who are most likely to benefit from mAbs treatment is currently challenging and limiting the impact of such therapies. To overcome this issue, and to fulfill the expectations of mAbs therapies, it is urgently required to develop proper culture models capable of faithfully reproducing the interactions between tumor and its surrounding native microenvironment (TME). Three-dimensional (3D) models which allow the assessment of the impact of drugs on tumors within its TME in a patient-specific context are promising avenues to progressively fill the gap between conventional 2D cultures and animal models, substantially contributing to the achievement of personalized medicine. This review aims to give a brief overview of the currently available 3D models, together with their specific exploitation for therapeutic mAbs testing, underlying advantages and current limitations to a broader use in preclinical oncology.

A Review of Methodologies for the Detection, Quantitation, and Localization of Free Cysteine in Recombinant Proteins: A Focus on Therapeutic Monoclonal Antibodies.

Free-cysteine residues in recombinant biotherapeutics such as monoclonal antibodies can arise from incorrect cellular processing of disulfide bonds during synthesis or by reduction of disulfide bonds during the harvest and purification stage of manufacture. Free cysteines can affect potency, induce aggregation, and decrease the stability of therapeutic proteins, and the levels and positions of free cysteines in proteins are closely monitored by both manufacturers and regulators to ensure safety and efficacy. This review summarizes the latest methodologies for the detection and quantification of free cysteines.

Recent Advances in Translational Pharmacokinetics and Pharmacodynamics Prediction of Therapeutic Antibodies Using Modeling and Simulation.

Therapeutic monoclonal antibodies (mAbs) have been a promising therapeutic approach for several diseases and a wide variety of mAbs are being evaluated in clinical trials. To accelerate clinical development and improve the probability of success, pharmacokinetics and pharmacodynamics (PKPD) in humans must be predicted before clinical trials can begin. Traditionally, empirical-approach-based PKPD prediction has been applied for a long time. Recently, modeling and simulation (M&S) methods have also become valuable for quantitatively predicting PKPD in humans. Although several models (e.g., the compartment model, Michaelis-Menten model, target-mediated drug disposition model, and physiologically based pharmacokinetic model) have been established and used to predict the PKPD of mAbs in humans, more complex mechanistic models, such as the quantitative systemics pharmacology model, have been recently developed. This review summarizes the recent advances and future direction of M&S-based approaches to the quantitative prediction of human PKPD for mAbs.

Glycan Profile Analysis of Engineered Trastuzumab with Rationally Added Glycosylation Sequons Presents Significantly Increased Glycan Complexity.

Protein aggregation constitutes a recurring complication in the manufacture and clinical use of therapeutic monoclonal antibodies (mAb) and mAb derivatives. Antibody aggregates can reduce production yield, cause immunogenic reactions, decrease the shelf-life of the pharmaceutical product and impair the capacity of the antibody monomer to bind to its cognate antigen. A common strategy to tackle protein aggregation involves the identification of surface-exposed aggregation-prone regions (APR) for replacement through protein engineering. It was shown that the insertion of N-glycosylation sequons on amino acids proximal to an aggregation-prone region can increase the physical stability of the protein by shielding the APR, thus preventing self-association of antibody monomers. We recently implemented this approach in the Fab region of full-size adalimumab and demonstrated that the thermodynamic stability of the Fab domain increases upon N-glycosite addition. Previous experimental data reported for this technique have lacked appropriate confirmation of glycan occupancy and structural characterization of the ensuing glycan profile. Herein, we mutated previously identified candidate positions on the Fab domain of Trastuzumab and employed tandem mass spectrometry to confirm attachment and obtain a detailed N-glycosylation profile of the mutants. The Trastuzumab glycomutants displayed a glycan profile with significantly higher structural heterogeneity compared to the HEK Trastuzumab antibody, which contains a single N-glycosylation site per heavy chain located in the CH2 domain of the Fc region. These findings suggest that Fab N-glycosites have higher accessibility to enzymes responsible for glycan maturation. Further, we have studied effects on additional glycosylation on protein stability via accelerated studies by following protein folding and aggregation propensities and observed that additional glycosylation indeed enhances physical stability and prevent protein aggregation. Our findings shed light into mAb glycobiology and potential implications in the application of this technique for the development of "biobetter" antibodies.

The Role of Fc Receptors on the Effectiveness of Therapeutic Monoclonal Antibodies.

Since the approval of the first monoclonal antibody (mAb) in 1986, a huge effort has been made to guarantee safety and efficacy of therapeutic mAbs. As of July 2021, 118 mAbs are approved for the European market for a broad range of clinical indications. In order to ensure clinical efficacy and safety aspects, (pre-)clinical experimental approaches evaluate the respective modes of action (MoA). In addition to antigen-specificity including binding affinity and -avidity, MoA comprise Fc-mediated effector functions such as antibody dependent cellular cytotoxicity (ADCC) and the closely related antibody dependent cellular phagocytosis (ADCP). For this reason, a variety of cell-based assays have been established investigating effector functions of therapeutic mAbs with different effector/target-cell combinations and several readouts including Fcγ receptor (FcγR)-mediated lysis, fluorescence, or luminescence. Optimized FcγR-mediated effector functions regarding clinical safety and efficacy are addressed with modification strategies such as point mutations, altered glycosylation patterns, combination of different Fc subclasses (cross isotypes), and Fc-truncation of the mAb. These strategies opened the field for a next generation of therapeutic mAbs. In conclusion, it is of major importance to consider FcγR-mediated effector functions for the efficacy of therapeutic mAbs.

MALDI-TOF mass spectrometry can distinguish immunofixation bands of the same isotype as monoclonal or biclonal proteins.

BACKGROUND: Plasma cell disorders (PCDs) are typically characterized by excessive production of a single immunoglobulin, defined as a monoclonal protein (M-protein). Some patients have more than one identifiable M-protein, termed biclonal. Traditional immunofixation electrophoresis (IFE) cannot distinguish if two bands of the same isotype represent biclonal proteins or M-proteins with some other feature. A novel assay using immunoenrichment coupled to matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (Mass-Fix) was applied to determine whether two bands of the same isotype represented (1) monomers and dimers of a single M-protein, (2) an M-protein plus a therapeutic monoclonal antibody (t-mAb), (3) an M-protein with light chain glycosylation, or (4) two distinct biclonal M-proteins. METHODS: Patient samples with two bands of the same isotype identified by IFE were enriched using nanobodies against IgG, IgA, IgM, or κ and λ light chains then analyzed by Mass-Fix. Light chain masses were used to differentiate IgGκ M-proteins from t-mAbs. Mass differences between peaks were calculated to identify N-glycosylation or matrix adducts. High-resolution mass spectrometry was used as a comparator method in a subset of samples. RESULTS: Eighty-one residual samples were collected. For IgA, 93% (n = 25) were identified as monoclonal. For IgG, 67% (n = 24) were monoclonal, and 33% (n = 12) were truly biclonal. Among the monoclonal IgGs, the second band represented a glycosylated form for 21% (n = 5), while 33% (n = 8) had masses consistent with a t-mAb. 44% (n = 8) of IgM samples were biclonal, and 56% (n = 10) were monoclonal, of which one was glycosylated. CONCLUSIONS: We demonstrate the utility of mass spectrometry in the characterization of multiple IFE bands of the same isotype. Improved reporting accuracy of M-proteins is useful for monitoring of patients with PCDs.

Targeting immunosuppression by TGF-ß1 for cancer immunotherapy.

The TGF-ß1 cytokine is a key mediator of many biological processes. Complex regulatory mechanisms are in place that allow one single molecule to exert so many distinct indispensable activities. The complexity of TGF-ß1 biology is further illustrated by the opposing dual roles it plays during cancer progression. Risks of toxicities combined with lack of convincing therapeutical efficacy explain at least in part why therapies targeting TGF-ß1 have lagged behind in past decades. However, recent successes of immunostimulatory antibodies for the immunotherapy of cancer and findings that TGF-ß1 activity associates with resistance to immunotherapeutic drugs have revived the field. In this review, we discuss the biology of TGF-ß1 with a special focus on its roles in regulating immune responses in the context of cancer. We describe the various therapeutic approaches available to inhibit TGF-ß signalling, and more recent findings that allow selective targeting of specific sources of TGF-ß activity, which may prove relevant to increase the efficacy and reduce the toxicity of cancer immunotherapy.

Evaluation of strategies for overcoming trifluoroacetic acid ionization suppression resulted in single-column intact level, middle-up, and bottom-up reversed-phase LC-MS analyses of antibody biopharmaceuticals.

A wide range of strategies for efficient chromatography and high MS sensitivity in reversed-phase LC-MS analysis of antibody biopharmaceuticals and their large derivates has been evaluated. They included replacing trifluoroacetic acid with alternative acidifiers, relevancy of elevated column temperature, use of dedicated stationary phases, and counteraction of the suppression effect of trifluoroacetic acid in electrospray ionization. At the column temperature of 60 °C, which significantly reduces in-column protein degradation, the BioResolve RP mAb Polyphenyl, BioShell IgG C4 columns performed best using mobile phases with full or partial replacement of trifluoroacetic acid with difluoroacetic acid in the analysis of intact antibodies. Similarly, 0.03% trifluoroacetic acid in combination with 0.07% formic acid is a good alternative in analyzing antibody chains at 60 °C. Collectively, the addition of 3% 1-butanol to the mobile phase acidified with 0.1% formic acid was the most efficient approach to simultaneously achieving good chromatographic separation and MS sensitivity for intact and reduced antibody biopharmaceuticals. Moreover, this mobile phase combined with the BioResolve RP mAb Polyphenyl column was subsequently demonstrated to provide excellent results for peptide mapping of antibody biopharmaceuticals fully comparable with those obtained using a state-of-the-art column for peptide separation, thus opening an avenue for a single-column multilevel analysis of these biotherapeutics.