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Alphabetical List of Abbreviations: Fab Fragment antigen-binding; Fc Fragment crystallizable; HMW High molecular weight; ∆HMW Difference between HMW species at stress temperature and 5°C controls; IgG Immunoglobulin G; mAbs Monoclonal antibodies; MV-V HH Multivalent V HH molecule with the format aC-L 1 -aC-L 1 -aD; NMR Nuclear magnetic resonance; scFv Single-chain fragment variable; SEC Size-exclusion chromatography; V HH Variable domain of Heavy chain of Heavy chain-only antibody.

One of the major product quality challenges for injectable biologics is controlling the amount of protein aggregates and particles present in the final drug product. This article focuses on particles in the submicron range (<2 μm). A cross-industry collaboration was undertaken to address some of the analytical gaps in measuring submicron particles (SMPs), developing best practices, and surveying the concentration of these particles present in 52 unique clinical and commercial protein therapeutics covering 62 dosage forms. Measured particle concentrations spanned a range of 4 orders of magnitude for nanoparticle tracking analysis and 3 orders of magnitude for resonant mass measurement. The particle concentrations determined by the 2 techniques differed significantly for both control and actual product. In addition, results suggest that these techniques exhibit higher variability compared to well-established subvisible particle characterization techniques (e.g., flow-imaging or light obscuration). Therefore, in their current states, nanoparticle tracking analysis and resonant mass measurement-based techniques can be used during product and process characterization, contributing information on the nature and propensity for formation of submicron particles and what is normal for the product, but may not be suitable for release or quality control testing. Evaluating the level of SMPs to which humans have been routinely exposed during the administration of several commercial and late-phase clinical products adds critical knowledge to our understanding of SMP levels that may be considered acceptable from a safety point of view. This article also discusses dependence of submicron particle size and concentration on the dosage form attributes such as physical state, primary packaging, dose strength, etc. To the best of our knowledge, this is the largest study ever conducted to characterize SMPs in late-phase and commercial products., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The success of biotherapeutic development heavily relies on establishing robust production platforms. During the manufacturing process, the protein is exposed to multiple stress conditions that can result in physical and chemical modifications. The modified proteins may raise safety and quality concerns depending on the nature of the modification. Therefore, the protein modifications potentially resulting from various process steps need to be characterized and controlled. This commentary brings together expertise and knowledge from biopharmaceutical scientists and discusses the various manufacturing process steps that could adversely impact the quality of drug substance (DS). The major process steps discussed here are commonly used in mAb production using mammalian cells. These include production cell culture, harvest, antibody capture by protein A, virus inactivation, polishing by ion-exchange chromatography, virus filtration, ultrafiltration-diafiltration, compounding followed by release testing, transportation and storage of final DS. Several of these process steps are relevant to protein DS production in general. The authors attempt to critically assess the level of risk in each of the DS processing steps, discuss strategies to control or mitigate protein modification in these steps, and recommend mitigation approaches including guidance on development studies that mimic the stress induced by the unit operations., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Insulin has a narrow therapeutic index, reflected in a small margin between a dose that achieves good glycemic control and one that causes hypoglycemia. Once injected, the clearance of exogenous insulin is invariant regardless of blood glucose, aggravating the potential to cause hypoglycemia. We sought to create a "smart" insulin, one that can alter insulin clearance and hence insulin action in response to blood glucose, mitigating risk for hypoglycemia. The approach added saccharide units to insulin to create insulin analogs with affinity for both the insulin receptor (IR) and mannose receptor C-type 1 (MR), which functions to clear endogenous mannosylated proteins, a principle used to endow insulin analogs with glucose responsivity. Iteration of these efforts culminated in the discovery of MK-2640, and its in vitro and in vivo preclinical properties are detailed in this report. In glucose clamp experiments conducted in healthy dogs, as plasma glucose was lowered stepwise from 280 mg/dL to 80 mg/dL, progressively more MK-2640 was cleared via MR, reducing by ∼30% its availability for binding to the IR. In dose escalations studies in diabetic minipigs, a higher therapeutic index for MK-2640 (threefold) was observed versus regular insulin (1.3-fold)., (© 2017 by the American Diabetes Association.)

The Biophorum Development Group (BPDG) is an industry-wide consortium enabling networking and sharing of best practices for the development of biopharmaceuticals. To gain a better understanding of current industry approaches for establishing biopharmaceutical drug product (DP) robustness, the BPDG-Formulation Point Share group conducted an intercompany collaboration exercise, which included a bench-marking survey and extensive group discussions around the scope, design, and execution of robustness studies. The results of this industry collaboration revealed several key common themes: (1) overall DP robustness is defined by both the formulation and the manufacturing process robustness; (2) robustness integrates the principles of quality by design (QbD); (3) DP robustness is an important factor in setting critical quality attribute control strategies and commercial specifications; (4) most companies employ robustness studies, along with prior knowledge, risk assessments, and statistics, to develop the DP design space; (5) studies are tailored to commercial development needs and the practices of each company. Three case studies further illustrate how a robustness study design for a biopharmaceutical DP balances experimental complexity, statistical power, scientific understanding, and risk assessment to provide the desired product and process knowledge. The BPDG-Formulation Point Share discusses identified industry challenges with regard to biopharmaceutical DP robustness and presents some recommendations for best practices., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Piggyback infusion has been widely used in the clinic with most applications in a nonconcurrent fashion for the purpose of administration convenience. In the present study, we demonstrated the application of concurrent piggyback to overcome challenges with intravenous administration of a salt-sensitive investigational protein. This setup consists of a syringe line containing drug admixture prepared in water-for-injection which is connected to a 0.9% sodium chloride line to keep vein open. Both lines are pump controlled and run concurrently at corresponding flow rate. The admixture compatibility study was conducted in 2 stages. In the first stage, admixture (concentration range from 0.05 to 2.0 mg/mL) was demonstrated to be compatible with water-for-injection and administration materials, such as intravenous bag, syringe, and syringe infusion line, for at least 24 h at room temperature. In the second stage, steady-state admixture concentration was demonstrated approximately 10 min after mixing even at the slowest syringe infusion rate. No loss of protein concentration was observed after reaching steady-state infusion. Subvisible particulates before and after piggybacking mixing are found well within the acceptable range., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Quantification and size distribution characterization of subvisible particles in parenteral biopharmaceutics, present as both proteinaceous and nonproteinaceous particles in the size range from 0.1 to 100 μm, are important for biopharmaceutical industry due to their potential safety and efficacy implications. Although a number of analytical techniques are available to count and size subvisible particles, characterization of particles ≤2 μm remains a significant challenge due to technical limitations of existing particle counting instruments. In this article, we demonstrate the ability of an optimized flow cytometry system to detect and quantify size distribution of subvisible particles without additional labeling that includes the critical submicron range in biopharmaceutical formulations. In addition, these qualitative and quantitative determinations are performed in a high-throughput manner using sample volumes that allow statistically significant evaluations. This approach can be used not only to ascertain the quality of therapeutic protein products but also to evaluate numerous conditions during the screening of drug candidates and their prospective formulations., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

In this study, an automated high-throughput relative chemical stability (RCS) assay was developed in which various therapeutic proteins were assessed to determine stability based on the resistance to denaturation post introduction to a chaotrope titration. Detection mechanisms of both intrinsic fluorescence and near UV circular dichroism (near-UV CD) are demonstrated. Assay robustness was investigated by comparing multiple independent assays and achieving r(2) values >0.95 for curve overlays. The complete reversibility of the assay was demonstrated by intrinsic fluorescence, near-UV CD, and biologic potency. To highlight the method utility, we compared the RCS assay with differential scanning calorimetry and dynamic scanning fluorimetry methodologies. Utilizing C1/2 values obtained from the RCS assay, formulation rank-ordering of 12 different mAb formulations was performed. The prediction of long-term stability on protein aggregation is obtained by demonstrating a good correlation with an r(2) of 0.83 between RCS and empirical aggregation propensity data. RCS promises to be an extremely useful tool to aid in candidate formulation development efforts based on the complete reversibility of the method to allow for multiple assessments without protein loss and the strong correlation between the C1/2 data obtained and accelerated stability under stressed conditions., (© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.)