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.

Considering "clonality": A regulatory perspective on the importance of the clonal derivation of mammalian cell banks in biopharmaceutical development.

There has been much recent focus on the regulatory emphasis and the relative importance surrounding clonal derivation of mammalian production cell lines used in the manufacture of recombinant DNA-derived biopharmaceuticals. This interest has led to an ongoing discussion between regulators and industry on how this topic is evaluated and the role it plays in the development of a new biopharmaceutical. Herein the authors describe that the clonal derivation of the production cell line is a factor with potential impact on product quality, and thus should not be considered separately from, but rather in the context of all elements comprising the control strategy necessary to support approval of a regulatory application. Considerations for how clonal derivation of cell banks and clonal variation thereof may be viewed during the lifecycle of a biopharmaceutical product is provided.

Cyberbiosecurity for Biopharmaceutical Products.

Cyberbiosecurity is an emerging discipline that addresses the unique vulnerabilities and threats that occur at the intersection of cyberspace and biotechnology. Advances in technology and manufacturing are increasing the relevance of cyberbiosecurity to the biopharmaceutical manufacturing community in the United States. Threats may be associated with the biopharmaceutical product itself or with the digital thread of manufacturing of biopharmaceuticals, including those that relate to supply chain and cyberphysical systems. Here, we offer an initial examination of these cyberbiosecurity threats as they stand today, as well as introductory steps toward paths for mitigation of cyberbiosecurity risk for a safer, more secure future.

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.

U.S. FDA Approval Summary: Nivolumab for Treatment of Unresectable or Metastatic Melanoma Following Progression on Ipilimumab.

On December 22, 2014, the FDA granted accelerated approval to nivolumab (OPDIVO; Bristol-Myers Squibb) for the treatment of patients with unresectable or metastatic melanoma and disease progression following ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor. Approval was based on a clinically meaningful, durable objective response rate (ORR) in a non-comparative analysis of 120 patients who received 3 mg/kg of nivolumab intravenously every 2 weeks with at least 6-month follow-up in an ongoing, randomized, open-label, active-controlled clinical trial. The ORR as assessed by a blinded independent review committee per RECIST v1.1 was 31.7% (95% confidence interval, 23.5-40.8). Ongoing responses were observed in 87% of responding patients, ranging from 2.6+ to 10+ months. In 13 patients, the response duration was 6 months or longer. The risks of nivolumab, including clinically significant immune-mediated adverse reactions (imARs), were assessed in 268 patients who received at least one dose of nivolumab. The FDA review considered whether the ORR and durations of responses were reasonably likely to predict clinical benefit, the adequacy of the safety database, and systematic approaches to the identification, description, and patient management for imARs in product labeling. Clin Cancer Res; 23(14); 3484-8. ©2017 AACR.

High relaxivity magnetic resonance imaging contrast agents. Part 1. Impact of single donor atom substitution on relaxivity of serum albumin-bound gadolinium complexes.

RATIONALE AND OBJECTIVES: The donor atoms that bind to gadolinium in contrast agents influence inner-sphere water exchange and electronic relaxation, both of which determine observed relaxivity. The effect of these molecular parameters on relaxivity is greatest when the contrast agent is protein bound. We sought to determine an optimal donor atom set to yield high relaxivity compounds. METHODS: A total of 38 gadolinium-1,4,7,10-tetraazacyclo-dodecane-N,N',N'',N'''-tetraacetato derivatives were prepared and relaxivity was determined in the presence and absence of human serum albumin as a function of temperature and magnetic field. Each compound had a common albumin-binding group and differed only by substitution of different donor groups at one of the macrocycle nitrogens. Oxygen-17 isotope relaxometry at 7.05 T was performed to estimate water exchange rates. RESULTS: Changing a single donor atom resulted in changes in water exchange rates ranging across 3 orders of magnitude. Donor groups increased water exchange rate in the order: phosphonate ∼ phenolate > α-substituted acetate > acetate > hydroxamate ∼ sulfonamide > amide ∼ pyridyl ∼ imidazole. Relaxivites at 0.47 and 1.4 T, 37°C, ranged from 12.3 to 55.6 mM(-1)s(-1) and from 8.3 to 32.6 mM(-1)s(-1) respectively. Optimal relaxivities were observed when the donor group was an α-substituted acetate. Electronic relaxation was slowest for the acetate derivatives as well. CONCLUSIONS: Water exchange dynamics and relaxivity can be predictably tuned by choice of donor atoms.

Europium luminescence of EF-hand helix-turn-helix chimeras: impact of pH and DNA-binding on europium coordination.

A series of Eu(III) metallopeptides, designed on the basis of the structural similarity of the helix-turn-helix and EF-hand motifs, have been studied by Eu(III) (7)F(0) --> (5)D(0) excitation spectroscopy. The impact of EF-hand ligand set differences on the hydration number and Eu(III) coordination environment are compared among the peptides. The conditional binding affinities were determined by Eu titration (P3, log K(a) = 6.0 +/- 0.4; P3W, log K(a) = 5.9 +/- 0.2; P5b, log K(a) = 5.3 +/- 0.1). Two similar coordination environments occur in each case, consistent with structural flexibility about the metal site. The coordination environments are consistent with 8- or 9-coordinate Eu(III), including six peptide-based ligands and two to three water molecules (P3, q = 1.9 +/- 0.2; P3W, q = 2.3 +/- 0.2; P4a, q = 1.9 +/- 0.3; P5b, q = 2.6 +/- 0.2). The Eu(III) (7)F(0) --> (5)D(0) excitation spectra are pH-dependent, as reported for several EF-hand proteins (oncomodulin, parvalbumin). A higher energy transition occurs at pH > 6, and has been assigned to deprotonation of coordinated water. The pK(a) leading to this new transition is dependent on Eu(III) Lewis acidity, which varies with the inner and outer sphere ligand set. The noncoordinating ninth position of the Eu-binding loop, which is poised to make second-sphere contacts to the coordinated water, stabilizes the deprotonated form of the coordinated solvent more effectively when it is Thr (P5b) than Asp (P3W). Upon DNA-binding by the metallopeptides, the pK(a) of the pH-dependent peak increases, but no new DNA-dependent transitions are observed. This indicates no DNA-based Eu(III) ligands are introduced, such as phosphate oxygen atoms of the DNA backbone. The hydration number decreases in the presence of DNA (P3W + DNA, q = 1.9 +/- 0.2; P5b + DNA, q = 1.7 +/- 0.2), indicating that DNA-binding by the metallopeptides organizes rather than compromises the Eu-binding site within the peptide.

Gadolinium-binding helix-turn-helix peptides: DNA-dependent MRI contrast agents.

A de novo designed gadolinium metallopeptide was found to be a very efficient relaxation agent, with 100% increase in relaxivity upon binding to DNA.

Sequence-selective DNA cleavage by a chimeric metallopeptide.

A chimeric metallopeptide derived from the sequences of two structurally superimposable motifs was designed as an artificial nuclease. Both DNA recognition and nuclease activity have been incorporated into a small peptide sequence. P3W, a 33-mer peptide comprising helices alpha2 and alpha3 from the engrailed homeodomain and the consensus EF-hand Ca-binding loop binds one equivalent of lanthanides or calcium and folds upon metal binding. The conditional formation constants (in the presence of 50 mM Tris) of P3W for Eu(III) (K(a) = (2.1 +/- 0.1) x 10(5) M(-1)) and Ce(IV) (K(a) = (2.6 +/- 0.1) x 10(5) M(-1)) are typical of isolated EF-hand peptides. Circular dichroism studies show that 1:1 CeP3W is 26% alpha-helical and EuP3W is up to 40% alpha-helical in the presence of excess metal. The predicted helicity of the folded peptide based on helix length and end effects is about 50%, showing the metallopeptides are significantly folded. EuP3W has considerably more secondary structure than our previously reported chimeras (Welch, J. T.; Sirish, M.; Lindstrom, K. M.; Franklin, S. J. Inorg. Chem. 2001, 40, 1982-1984). Eu(III)P3W and Ce(IV)P3W nick supercoiled DNA at pH 6.9, although EuP3W is more active at pH 8. CeP3W cleaves linearized, duplex DNA as well as supercoiled plasmid. The cleavage of a 5'-(32)P-labeled 121-mer DNA fragment was followed by polyacrylamide gel electrophoresis. The cleavage products are 3'-OPO(3) termini exclusively, suggesting a regioselective or multistep mechanism. In contrast, uncomplexed Ce(IV) and Eu(III) ions produce both 3'-OPO(3) and 3'-OH, and no evidence of 4'-oxidative cleavage termini with either metal. The complementary 3'-(32)P-labeled oligonucleotide experiment also showed both 5'-OPO(3) and 5'-OH termini were produced by the free ions, whereas CeP3W produces only 5'-OPO(3) termini. In addition to apparent regioselectivity, the metallopeptides cut DNA with modest sequence discrimination, which suggests that the HTH motif binds DNA as a folded domain and thus cleaves selected sequences. The de novo artificial nuclease LnP3W represents the first small, underivatized peptide that is both active as a nuclease and sequence selective.

Lanthanide-binding helix-turn-helix peptides: solution structure of a designed metallonuclease.

A designed lanthanide-binding chimeric peptide based on the strikingly similar geometries of the EF-hand and helix-turn-helix (HTH) motifs was investigated by NMR and CD spectroscopy and found to retain the same overall solution structure of the parental motifs. CD spectroscopy showed that the 33-mer peptide P3W folds on binding lanthanides, with an increase in alpha-helicity from 20% in the absence of metal to 38% and 35% in the presence of excess Eu(III) and La(III) ions, respectively. The conditional binding affinities of P3W for La(III) (5.9 +/- 0.3 microM) and for Eu(III) (6.2 +/- 0.3 microM) (pH 7.8, 5 mM Tris) were determined by tryptophan fluorescence titration. The La(III) complex of peptide P3, which differs from P3W by only one Trp-to-His substitution, has much less signal dispersion in the proton NMR spectra than LaP3W, indicating that the Trp residue is a critical hydrophobic anchor for maintaining a well-folded helix-turn-helix structure. A chemical-shift index analysis indicates the metallopeptide has a helix-loop-helix secondary structure. A structure calculated by using nuclear Overhauser effect and other NMR constraints reveals that P3W not only has a tightly folded metal-binding loop but also retains the alpha-alpha corner supersecondary structure of the parental motifs. Although the solution structure is undefined at both the N and C termini, the NMR structure confirms the successful incorporation of a metal-binding loop into a HTH sequence.