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Nucleic acid (NA)-based biopharmaceuticals have emerged as promising therapeutic modalities. NA therapeutics are a diverse class of RNA and DNA and include antisense oligonucleotides, siRNA, miRNA, mRNA, small activating RNA, and gene therapies. Meanwhile, NA therapeutics have posed significant stability and delivery challenges and are expensive. This article discusses the challenges and opportunities for achieving stable formulations of NAs with novel drug delivery systems (DDSs). Here we review the current progress in the stability issues and the significance of novel DDSs associated with NA-based biopharmaceuticals, as well as mRNA vaccines. We also highlight the European Medicines Agency (EMA) and US Food and Drug Administration (FDA)-approved NA-based therapeutics with their formulation profiles. NA therapeutics could impact future markets if the remaining challenges and requirements are addressed. Regardless of the limited information available for NA therapeutics, reviewing and collating the relevant facts and figures generates a precious resource for formulation experts familiar with the NA therapeutics' stability profile, their delivery challenges, and regulatory acceptance. [ABSTRACT FROM AUTHOR]

Peptide C-terminal N-alkyl amides have gained more attention over the past decade due to their biological properties, including improved pharmacokinetic and pharmacodynamic profiles. However, the synthesis of this type of peptide on solid phase by current available methods can be challenging. Here we report a convenient method to synthesize peptide C-terminal N-alkyl amides using the well-known Fukuyama N-alkylation reaction on a standard resin commonly used for the synthesis of peptide C-terminal primary amides, the peptide amide linker-polyethylene glycol-polystyrene (PAL-PEG-PS) resin. The alkylation and oNBS deprotection were conducted under basic conditions and were therefore compatible with this acid labile resin. The alkylation reaction was very efficient on this resin with a number of different alkyl iodides or bromides, and the synthesis of model enkephalin N-alkyl amide analogs using this method gave consistently high yields and purities, demonstrating the applicability of this methodology. The synthesis of N-alkyl amides was more difficult on a Rink amide resin, especially the coupling of the first amino acid to the N-alkyl amine, resulting in lower yields for loading the first amino acid onto the resin. This method can be widely applied in the synthesis of peptide N-alkyl amides. © 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 96: 715-722, 2011. [ABSTRACT FROM AUTHOR]

[Display omitted] • Prefilled dual chamber devices (DCDs) as emerging drug delivery system for biopharmaceuticals. • Composition and manufacturing challenges for prefilled DCDs. • Current global market scenario for prefilled DCDs. • ISO standards and CCI quality requirements for prefilled DCDs. • Role of extractables and leachables on the quality of prefilled DCDs. Prefilled dual chamber devices (DCDs) are combination products containing freeze-dried drug and diluent in two separate chambers of the device. DCDs provide high stability and convenience to patients and doctors, thus significantly improving product quality, patient compliance and market competitiveness. DCDs should also provide seal integrity, sterility and compatibility with biopharmaceuticals and avoid leachability and needle stick injuries. DCDs are promising alternatives to traditional containers or devices for biopharmaceuticals. The regulatory and medical practice to choose plastic DCDs as better alternatives over well-established glass syringes will be addressed here. The impact and major issues during processing, manufacturing, and storage of DCDs are also highlighted. Further discussion clears its business potential, composition, stability testing, and quality standard requirements to deal with market competition. It also covers major role of extractables and leachables in storage stability of the product. [ABSTRACT FROM AUTHOR]

[Display omitted] Adequate stabilization is essential for marketed protein-based biopharmaceutical formulations to withstand the various stresses that can be exerted during the pre- and post-manufacturing processes. Therefore, a suitable choice of excipient is a significant step in the manufacturing of such delicate products. Histidine, an essential amino acid, has been extensively used in protein-based biopharmaceutical formulations. The physicochemical properties of histidine are unique among amino acids and could afford multifaceted benefits to protein-based biopharmaceutical formulations. With a pKa of approximately 6.0 at the side chain, histidine has been primarily used as a buffering agent, especially for pH 5.5–6.5. Additionally, histidine exhibited several affirmative properties similar to those of carbohydrates (e.g., sucrose and trehalose) and could therefore be considered to be an alternative approach to established protein-based formulation strategies. The current review describes the general physicochemical properties of histidine, lists all commercial histidine-containing protein-based biopharmaceutical products, and discusses a brief outline of the existing research focused on the versatile applications of histidine, which can act as a buffering agent, stabilizer, cryo-/lyo-protectant, antioxidant, viscosity reducer, and solubilizing agent. The interaction between histidine and proteins in protein-based biopharmaceutical formulations, such as the Donnan effect during diafiltration of monoclonal antibody solutions and the degradation of polysorbates in histidine buffer, has also been discussed. As the first review of histidine in protein biopharmaceuticals, it helps to deepen our understanding of the opportunities and challenges associated with histidine as an excipient for protein-based biopharmaceutical formulations. [ABSTRACT FROM AUTHOR]

Capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) has long been proven to have excellent performance in the analysis and characterization of therapeutic proteins. However, it is rarely used for the detection of low-molecular-weight proteins or peptides. Our research has proved the ability of CE-SDS to characterize the purity of low-molecular-weight proteins (i.e., <10 kDa) and even polypeptides. In this article, insulin glargine was used as a model protein, and CE-SDS was used to analyze the samples damaged by heating and light exposure. The monomers, dimers, and trimers of insulin glargine were effectively separated, and the results of the mass spectrometry also confirmed the existence of two kinds of insulin aggregates. For comparison, the size-exclusion high-performance liquid chromatography (SE-HPLC) only showed a single aggregate peak. In addition, the denaturation conditions caused only the covalent aggregates to appear in the CE-SDS analysis. These advantages also make CE-SDS an excellent supplementary technology to the traditional SE-HPLC, providing biopharmaceutical analysts with more information. • Low-molecular-weight protein aggregation was first characterized by CE-SDS. • Insulin glargine aggregation was compared between CE-SDS and SE-HPLC. • CE-SDS achieved higher resolution and distinguished covalent from noncovalent aggregates. [ABSTRACT FROM AUTHOR]

For monoclonal antibody (mAb) drugs, the 'me-too' drug is a pharmacologically active compound that is structurally similar to the first-in-class drugs, acting on the same target and is used for the same therapeutic purposes, but it may differ in drug-drug interactions and adverse drug reactions. Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) has been widely used for quality evaluation of mAb drugs. The properties of the detected substances can interfere with the credibility and accuracy of the method. In the routine comparison analysis for both innovator rituximab and 'me-too' drug zuberitamab samples, an uncommon artifact related to the heavy chain (HC) of zuberitamab was observed in reducing CE-SDS and interfered with our identification of the purity of samples. In this work, the overall hydrophobicity of the HCs of rituximab, zuberitamab, and several other common mAbs was characterized and determined by reversed-phase high-performance liquid chromatography. Additionally, the local hydrophobicity and surface charge were compared using Expasy ProtScale and PyMOL software simulations. We concluded that noncovalent protein aggregation can be related to strong hydrophobicity and low electrostatic repulsion of local amino acid regions, which complicates drug quality control. These findings shed light on the relationship between protein aggregation and the local hydrophobicity region, and broaden the way to analyze the detection 'artifacts' in reducing CE-SDS studies of therapeutic proteins. • An uncommon artifact was generated during CE-SDS analysis of zuberitamab. • Structures were compared between zuberitamab and other mAbs. • Strong local hydrophobicity and low electrostatic repulsion were the root causes. [ABSTRACT FROM AUTHOR]

Arodyn (Ac[Phe¹,²,³,Arg⁴,D-Ala⁸]Dyn A(1-11)NH₂) is an acetylated dynorphin A (Dyn A) analog that is a potent and selective κ opioid receptor antagonist (Bennett et al., J Med Chem 2002, 45, 5617), and its analog [NMePhe¹]arodyn shows even higher affinity and selectivity for κ opioid receptors (Bennett et al., J Pept Res 2005, 65, 322). However, the latter compound is prone to deletion of the Ac-NMePhe moiety from the N-terminus of the peptide during acidic cleavage as described in the accompanying paper. Several stable analogs of [NMePhe¹]arodyn and [NMePhe¹,Trp³]arodyn where the acetyl group was substituted with a heteroatom-containing group were evaluated for their opioid receptor affinity, selectivity, and efficacy. Methoxycarbonyl derivatives exhibited the highest κ opioid receptor affinity among the analogs. Additional [CH₃OCO-NMePhe¹]arodyn analogs where position 3 was substituted with other aromatic or nonaromatic residues were also evaluated for κ receptor affinity, selectivity, and efficacy. [CH₃OCO-NMePhe¹]arodyn has similar κ opioid receptor affinity as [NMePhe¹]arodyn, retains high κ opioid receptor selectivity, and is a potent κ opioid receptor antagonist.

Peptides containing N-methylamino acids can exhibit improved pharmacodynamic and pharmacokinetic profiles compared to nonmethylated peptides, and therefore interest in these N-methylated peptides has been increasing in recent years. Arodyn (Ac[Phe¹,²,³,Arg⁴,D-Ala⁸]Dyn A(1-11)NH₂) is an acetylated dynorphin A(Dyn A) analog that is a potent and selective κ opioid receptor antagonist (Bennett et al., J Med Chem 2002, 45, 5617), and its analog [NMePhe¹]arodyn shows even higher affinity and selectivity for κ opioid receptors (Bennett et al., J Pept Res 2005, 65, 322). During the synthesis of [NMePhe¹]arodyn analogs, the arodyn-(2-11) derivatives were obtained as major products. Analysis indicated that Ac-NMePhe was lost from the completed peptide sequence during acidic cleavage of the peptides from the resin and that the acetyl group played an important role in this side reaction. Different cleavage conditions were evaluated to minimize this side reaction and maximize the yield of pure [NMePhe¹]arodyn analogs. Modifications to the N-terminus of the peptides to prevent the side reaction were also explored. The incorporation of a heteroatom-containing group such as methoxycarbonyl as the N-terminal functionality prevented this side reaction, while the incorporation of a bulky acyl group could not. Substituting NMePhe with the conformationally constrained analog Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) also prevented the side reaction.

Dynorphin A (Dyn A) is an endogenous ligand for kappa opioid receptors. To restrict the conformational mobility, we synthesized several cyclic Dyn A-(1-11)NH(2) analogues on solid phase utilizing ring-closing metathesis (RCM) between the side chains of allylglycine (AllGly) residues incorporated in positions 2, 5, and/or 8. Cyclizations between the side chains of AllGly gave reasonable yields (56-74%) of all of the desired cyclic peptides. Both the cis and trans isomers were obtained for all of the cyclic peptides, with the ratio of cis to trans isomers depending on the position and stereochemistry of the AllGly. Most of the cyclic Dyn A-(1-11)NH(2) analogues examined exhibit low nanomolar binding affinity for kappa opioid receptors (K(i) = 0.84-11 nM). In two of the three cases, the configuration of the double bond has a significant influence on the opioid receptor affinities and agonist potency. All of the peptides inhibited adenylyl cyclase activity in a concentration-dependent manner with full or close to full agonist activity. These potent Dyn A analogues are the first ones cyclized by RCM.