The Mannose in the Mirror: A Reflection on the Pharmacokinetic Impact of High Mannose Glycans of Monoclonal Antibodies in Biosimilar Development.

Biosimilar development has a well-documented foundation of product quality and extensive comparative analytics providing the bulk of the "totality of the evidence" that a proposed product is biosimilar to its reference product. This work provides a retrospective evaluation of a single critical quality attribute-high mannose glycans for monoclonal antibody biosimilars. Given the well-established conclusion that high mannose glycans can impact pharmacokinetic (PK) profile, we performed a retrospective evaluation of 21 monoclonal antibody biosimilar programs (those licensed before April 2022), their levels of glycans, and the methods used to study them. We provide herein a summary of the methods used and their relative performance. We also present a subset analysis for seven biosimilar products with levels of high mannose that differ from the corresponding reference product (and where other differences in quality attributes between the two that may influence PK profile were not observed or considered minor) and compared the PK profiles. Critically, this analysis has demonstrated that the measurement of glycan profiles is highly precise, reproducible within and across programs, and can detect differences in mannose levels, even those that do not impact PK. These results provide support that analytics rather than pharmacokinetic data may be sufficient to predict whether differences within a certain magnitude of this attribute are likely to impact PK. This work enhances the Agency's understanding of this issue allowing for better understanding of challenges faced by the biotechnology industry developing biosimilars.

The role of immune tolerance induction in restoration of the efficacy of ERT in Pompe disease.

Pompe disease is a lysosomal storage disorder caused by deficiency in the enzyme acid α-glucosidase (GAA). Pompe disease is characterized by the accumulation of glycogen, predominantly in muscle tissue, leading to progressive muscle weakness, loss of motor, respiratory, and, in the infantile-onset form, cardiac function. Disease progression is highly variable depending on phenotype, but premature death due to respiratory complications occurs in most patients. Beginning in 2006, approved alglucosidase alfa enzyme replacement therapies [recombinant human (rh) GAA] have been available to treat Pompe patients. Treatment of classic infantile-onset patients, who manifest the severest form of the disease, with alglucosidase alfa (Myozyme®) has led to extended survival and an evolving understanding of the pathophysiology and course of the disease. Moreover, such treatment has brought to light the role of the immune response in abrogating the efficacy of rhGAA in classic infantile-onset patients with severe genetic mutations. Thus, optimization of treatment for such patients includes development and utilization of strategies to prevent or eliminate immune responses, including modulating the immune system (prophylactic and therapeutic immune tolerance induction regimens) and engineering the enzyme to be less immunogenic and more effective. Future research is also critical for evaluating and mitigating novel disease-associated pathologies uncovered by prolonged survival of infantile-onset patients including development of novel therapeutics, and for protein design strategies to increase delivery of enzyme replacement therapy to critical target tissues. Such efforts would be greatly bolstered by further development of predictive animal models and biomarkers to facilitate clinical trials and patient management. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.

PDA Biosimilars Workshop Report (September 27-28, 2018)-Getting It Right the First Time for Biosimilar Marketing Applications.

This workshop report summarizes the presentations, the breakout session outcomes, and the speaker panel discussions from the PDA Biosimilars Workshop held September 27-28, 2018, in Washington, DC. This format was deliberately selected for the workshop with the expectation of delivering a post-workshop paper on current best practices and existing challenges for sponsors. The event, co-chaired by Dr. Stephan Krause (AstraZeneca Biologics) and Dr. Emanuela Lacana (CDER/FDA), was attended by 140 agency and industry representatives. The workshop was separated into three major sessions P1: Regulatory Perspective, P2: Challenges in Biosimilar Development, and P3: Demonstrating Analytical Similarity. Each of the three sessions started with agency and industry presentations. Participants then split into two concurrent roundtable discussion groups to hear the answers to questions that had been provided to all participants one week prior to the event. The sessions were recorded. This paper provides consolidated answers to specific case studies for current challenges to sponsors and agencies. In addition, the panel discussion notes following each breakout roundtable session, as well as brief talk summaries of all speakers, are provided. The first session explored the challenges encountered with submission of biosimilar marketing applications from the perspectives of regulatory agencies. Expectations for a successful submission of the chemistry, manufacturing, and controls (CMC) information were described. The second session addressed high-level technical challenges and how to avoid pitfalls frequently encountered during biosimilar candidate development, including data quality expectations, creation of the final control strategy, and strategic choices necessary for candidate selection and development. Both regulatory perspectives and industry experience were shared. The last session explored the use of statistical tools to provide meaningful contributions to the demonstration of analytical similarity. The presentations highlighted common issues and practical challenges that arise during the application of statistical tools.LAY ABSTRACT: Significant challenges are still-remaining for sponsors and agencies to successfully develop and license Biosimilars. A Biosimilars Workshop was therefore held on 27-28 September 2018 in Washington, DC, to find practical solutions to the remaining challenges. The workshop planning committee with members from industry and agencies prepared specific case studies focused on some of most difficult situations. The workshop was separated into three major sessions (P1 - Regulatory Perspective; P2 - Challenges in Biosimilar Development; P3 - Demonstrating Analytical Similarity) and each session attempted to provide practical solutions to the relevant case studies. This first session explored the challenges encountered with submission of biosimilar marketing applications from the regulatory agencies' perspectives. Expectations for a successful submission of the CMC information were described. The second session addressed high-level technical challenges frequently encountered during biosimilar candidate development, including data quality expectations, the creation of the final control strategy, and strategic choices necessary for candidate selection and development. The last session explored the use of statistical tools to provide meaningful contributions to the demonstration of analytical similarity and practical challenges that arise during the application of statistical tools.

Apoptosis-linked gene 2-deficient mice exhibit normal T-cell development and function.

The apoptosis-linked gene product, ALG-2, is a member of the family of intracellular Ca(2+)-binding proteins and a part of the apoptotic machinery controlled by T-cell receptor (TCR), Fas, and glucocorticoid signals. To explore the physiologic function of ALG-2 in T-cell development and function, we generated mice harboring a null mutation in the alg-2 gene. The alg-2 null mutant mice were viable and fertile and showed neither gross developmental abnormality nor immune dysfunction. Analyses of apoptotic responses of ALG-2-deficient T cells demonstrated that ALG-2 deficiency failed to block apoptosis induced by TCR, Fas, or dexamethasone signals. These findings indicate that ALG-2 is physiologically dispensable for apoptotic responses induced by the above signaling pathways and suggest that other functionally redundant proteins might exist in mammalian cells.

Advancing Product Quality: a Summary of the Inaugural FDA/PQRI Conference.

On September 16 and 17, 2014, the Food and Drug Administration (FDA) and Product Quality Research Institute (PQRI) inaugurated their Conference on Evolving Product Quality. The Conference is conceived as an annual forum in which scientists from regulatory agencies, industry, and academia may exchange viewpoints and work together to advance pharmaceutical quality. This Conference Summary Report highlights key topics of this conference, including (1) risk-based approaches to pharmaceutical development, manufacturing, regulatory assessment, and post-approval changes; (2) FDA-proposed quality metrics for products, facilities, and quality management systems; (3) performance-based quality assessment and clinically relevant specifications; (4) recent developments and implementation of continuous manufacturing processes, question-based review, and European Medicines Agency (EMA)-FDA pilot for Quality-by-Design (QbD) applications; and (5) breakthrough therapies, biosimilars, and international harmonization, focusing on ICH M7 and Q3D guidelines. The second FDA/PQRI conference on advancing product quality is planned for October 5-7, 2015.

Neuronal apoptosis is accompanied by amyloid beta-protein accumulation in the endoplasmic reticulum.

A series of evidences suggests that enhanced susceptibility to programmed cell death (PCD) is a major pathogenetic factor in Alzheimer's disease (AD). We investigated this issue, analyzing amyloid beta-protein (A beta) production in a model of neuronal PCD, induced by staurosporine in a murine neuroblastoma cell line. When PCD was induced, a 280-290% secreted A beta occurred, in spite of a 20% metabolism and an unchanged A betaPP expression. The increased intracellular A beta reactivity largely colocalized with a marker of ER. Inhibition of caspases blocked the cleavage at the C-terminus of beta PP, but only partially rescued A beta overproduction caused by staurosporine treatment. Our findings suggest that PCD fosters the physiological pathways of A beta production characteristic of neuronal cells, and they confirm the theory that unbalance of PCD is a central event in AD pathogenesis. Moreover, our data indicate that still unidentified cellular mechanisms, other than caspases activation, are responsible of the specific alteration of A betaPP processing during PCD.

Cloning and characterization of a protein kinase A anchoring protein (AKAP)-related protein that interacts with and regulates sphingosine kinase 1 activity.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that has novel dual actions. S1P is the ligand for a family of G protein-coupled receptors known as S1PRs that mediate various physiological functions. Growth factors rapidly activate sphingosine kinase type 1 (SPHK1) resulting in phosphorylation of sphingosine to form S1P, which plays important roles in cell growth regulation and protection from apoptosis. However, little is known of the mechanism(s) by which SPHK activity is regulated. Using a yeast two-hybrid screening approach, we cloned a 3-kb cDNA encoding a SPHK1-interacting protein (SKIP). BLAST analysis revealed that SKIP corresponded to the C-terminal region of a larger ( approximately 7 kb) cDNA that encoded a protein with a high degree of similarity to a family of protein kinase A anchor proteins (AKAP). In confirmation of the yeast two-hybrid assay, glutathione S-transferase (GST)-SPHK1 specifically pulled down SKIP, whereas GST did not. Moreover, immunoprecipitation of in vitro translated SPHK1 and SKIP revealed that SKIP and SPHK1 are tightly associated. Furthermore, SKIP overexpression in NIH 3T3 fibroblasts reduced SPHK1 activity and interfered with its biological functions. The apoptotic-sparing effect of SPHK1 against serum deprivation was reduced when co-transfected with SKIP. In addition, SPHK1-enhanced cell proliferation was also abolished by SKIP, with a corresponding decrease in activation of ERK. Taken together, these results indicate that SKIP is a novel protein likely to play a regulatory role in the modulation of SPHK1 activity.

The G protein-coupled receptor GPR4 suppresses ERK activation in a ligand-independent manner.

The lysophospholipids, lysophosphatidic acid, sphingosine-1-phosphate, and sphingosylphosphorylcholine (SPC), are bioactive lipid molecules that regulate diverse biological processes. Although the specific G protein-coupled receptors for lysophosphatidic acid and sphingosine-1-phosphate have been well-characterized, much less is known of the SPC receptors. It has been reported that ovarian cancer G protein-coupled receptor 1 (OGR1) is a high affinity receptor for SPC, and its closely related homologue GPR4 is a high affinity receptor for SPC with low affinity for lysophosphatidylcholine (LPC). However, in a functional assay to examine the specificity of ligand binding, we found that neither SPC nor LPC, or other related lysophospholipids, induced internalization of GPR4 from the plasma membrane. In agreement, these lysolipids also did not induce translocation of beta-arrestin2-GFP from the cytosol to the plasma membrane in GPR4 expressing cells. However, when these cells were cotransfected with G protein-coupled receptor kinase 2, in the absence of added ligands, beta-arrestin2-GFP accumulated in cytoplasmic vesicles, reminiscent of vesicular labeling usually observed after agonist stimulation of GPCRs. In addition, neither SPC nor LPC stimulated the binding of GTPgammaS to membranes prepared from GPR4 expressing cells and did not activate ERK1/2. Surprisingly, enforced expression of GPR4 inhibited activation of ERK1/2 induced by several stimuli, including SPC, sphingosine-1-phosphate, and even EGF. Collectively, our results suggest that SPC and LPC are not the ligands for GPR4 and that this receptor may constitutively inhibit ERK1/2 activation.

A LAT mutation that inhibits T cell development yet induces lymphoproliferation.

Mice homozygous for a single tyrosine mutation in LAT (linker for activation of T cells) exhibited an early block in T cell maturation but later developed a polyclonal lymphoproliferative disorder and signs of autoimmune disease. T cell antigen receptor (TCR)-induced activation of phospholipase C-gamma1 (PLC-gamma1) and of nuclear factor of activated T cells, calcium influx, interleukin-2 production, and cell death were reduced or abrogated in T cells from LAT mutant mice. In contrast, TCR-induced Erk activation was intact. These results identify a critical role for integrated PLC-gamma1 and Ras-Erk signaling through LAT in T cell development and homeostasis.