Stefan G. Koenig

Targeted drug delivery through the traceless release of tertiary and heteroaryl amines from antibody–drug conjugates

The reversible attachment of a small-molecule drug to a carrier for targeted delivery can improve pharmacokinetics and the therapeutic index. Previous studies have reported the delivery of molecules that contain primary and secondary amines via an amide or carbamate bond; however, the ability to employ tertiary-amine-containing bioactive molecules has been elusive. Here we describe a bioreversible linkage based on a quaternary ammonium that can be used to connect a broad array of tertiary and heteroaryl amines to a carrier protein. Using a concise, protecting-group-free synthesis we demonstrate the chemoselective modification of 12 complex molecules that contain a range of reactive functional groups. We also show the utility of this connection with both protease-cleavable and reductively cleavable antibody-drug conjugates that were effective and stable in vitro and in vivo. Studies with a tertiary-amine-containing antibiotic show that the resulting antibody-antibiotic conjugate provided appropriate stability and release characteristics and led to an unexpected improvement in activity over the conjugates previously connected via a carbamate.

A deeper shade of green: inspiring sustainable drug manufacturing

Green and sustainable drug manufacturing go hand in hand with forward-looking visions seeking to balance the long-term sustainability of business, society, and the environment. However, a lack of harmonization among available metrics has inhibited opportunities for green chemistry in industry. Moreover, inconsistent starting points for analysis and neglected complexities for diverse manufacturing processes have made developing objective goals a challenge. Herein we put forward a practical strategy to overcome these barriers using data from in-depth analysis of 46 drug manufacturing processes from nine large pharmaceutical firms, and propose the Green Aspiration Level as metric of choice to enable the critically needed consistency in smart green manufacturing goals. In addition, we quantify the importance of green chemistry in the often overlooked, yet enormously impactful, outsourced portion of the supply chain, and introduce the Green Scorecard as a value added sustainability communication tool.

Development of a tripartite solvent blend for sustainable chromatography

A three-component solvent system (isopropyl acetate, methanol, and heptane) was designed to empower routine chromatographic separation of a wide range of compounds, from polar to nonpolar molecules, without the need for undesired chlorinated solvent. This system was evaluated with a pharmaceutically-relevant set of compounds via practical silica gel chromatographic purification, making the solvent blend an attractive sustainable chromatography solution for drug discovery efforts.

Inspiring process innovation via an improved green manufacturing metric: iGAL

Following our goal to devise a unified green chemistry metric that inspires innovation in sustainable drug manufacturing across the pharmaceutical industry, we herein disclose joint efforts by IQ, the ACS GCI PR and academia, leading to the significantly improved ‘innovation Green Aspiration Level’ (iGAL) methodology. Backed by the statistical analysis of 64 drug manufacturing processes encompassing 703 steps across 12 companies, we find that iGAL affords an excellent proxy for molecular complexity and presents a valuable molecular weight-based ‘fixed’ goal. iGAL thereby accurately captures the impact of green process inventiveness and improvements, making it a useful innovation-driven green metric. We conclude by introducing the comprehensive, yet easy-to-use and readily adaptable Green Chemistry Innovation Scorecard web calculator, whose graphical output clearly and effectively illustrates the impact of innovation on waste reduction during drug manufacture.

Introduction of a process mass intensity metric for biologics

Biopharmaceuticals (or biologics), large molecule therapeutics typically produced using biotechnology, are a rapidly growing segment of the pharmaceutical market. As such, the environmental footprint of the production of these molecules is coming under scrutiny from various stakeholders such as healthcare providers, investors, and even employees. Process mass intensity (PMI), originally adopted for small molecules by the Green Chemistry Institute Pharmaceutical Roundtable, is a simple metric that can also be applied to evaluate the process efficiency of biopharmaceutical production. PMI for biologics is defined as the total mass input in kg of water, raw materials and consumables, required to make 1 kg of active pharmaceutical ingredient. Six large pharmaceutical companies participated in a benchmarking exercise to calculate the PMI for monoclonal antibody (mAb) production. On average, 7700 kg of input is required to produce 1 kg of mAb. Over 90% of the mass is due to water use, highlighting the water-intensive nature of biologics production.

Highly Efficient Synthesis of a Staphylococcus aureus Targeting Payload to Enable the First Antibody-Antibiotic Conjugate

A practical synthesis of the complex payload for an anti-Staphylococcus aureus THIOMABTM antibody-antibiotic conjugate (TAC) is described. The route takes advantage of a delicate oxidative condensation, achieved using a semi-continuous flow procedure. It allows for the generation of kilogram quantities of a key intermediate to enable a mild nucleophilic aromatic substitution to the tertiary amine free drug. The linker component is introduced as a benzylic chloride, which allows formation of the quaternary ammonium salt linker-drug. This chemical process surmounts numerous synthetic challenges and navigates deeply colored and unstable compounds to support clinical studies to counter S. aureus bacterial infections.

A call for industry to embrace green biopharma

VOLUME 34 NUMBER 3 MARCH 2016 NATURE BIOTECHNOLOGY To the Editor: Awareness and understanding about environmental risks have grown considerably in recent years, resulting in public policies and private sector efforts to encourage further development of environmentally sustainable business practices. For companies, the business benefits of environmentally conscious decision-making include reducing regulatory risks and operational costs, better management of scarce resources, and improving corporate reputation1. Pharmaceutical companies embarked on this journey over a decade ago with the incorporation of ‘Green Chemistry and Engineering Principles’ into small-molecule drug development and production through the American Chemical Society (ACS; Washington, DC, USA) Green Chemistry Institute Pharmaceutical Roundtable (GCIPR)2,3. As large-molecule drugs (biologics or biopharmaceuticals) have grown in importance, so too has the need to employ cost-efficient, environmentally sustainable production methods for these drugs. Here we argue for the need to adapt the Green Chemistry and Engineering Principles to biologics and to develop appropriate environmental metrics as a means to accelerate the greening of the biopharmaceutical sector. We hope that the examples provided in this article will encourage other companies to further develop and implement the tenets of what we term ‘green biopharma’. Biopharmaceuticals, for the purpose of this discussion, are biological compounds produced by fermentation as opposed to chemical synthesis, and include antibodies, proteins and some vaccines. Large amounts of water are consumed during manufacturing, estimated at millions of liters annually (3,000–7,000 kg of water per kg of drug produced on the basis of 30,000 kg of drug produced each year; when cleaning operations are included these numbers can increase by an order of magnitude)4–6. As the world’s population continues to grow and become richer, demand for this irreplaceable resource increases. Indeed, in 2010, the United Nations passed a resolution recognizing the right of every human to have access to clean water and sanitation7. Biopharmaceutical companies have recognized the impact their processes have on local water quality and availability, and many have committed themselves to reducing their water consumption8–11. In addition, companies generate substantial solid waste from the use of consumables such as filters, bags and chromatography resins; at the same time, large amounts of energy are expended to maintain cleanroom environments and to operate equipment. There remains, however, a major opportunity to further reduce the environmental impact of biologics manufacturing and mitigate risk to the industry. To address these opportunities, Genentech (S. San Francisco, CA), a member of the Roche group, created an internal green biopharma program and helped establish the Biopharma Focus Group (BFG) within the ACS GCIPR to bring the approach to the wider industry. We define green biopharma as the design, development and implementation of biological and chemical products and processes that reduce or eliminate our impact on human health and the environment. In 2011, a cross-disciplinary Green BioPharma Steering Committee was created at Genentech, composed of scientists and engineers from largeand small-molecule research, biologics technical development and engineering, manufacturing sciences, quality groups, and environment, health and safety. This committee manages a project portfolio to further the aims of the program. It is built around four strategic focus areas: for chemicals, the focus is to decrease the company’s chemical footprint by using fewer hazardous chemicals and reducing the amount of hazardous waste generated; for materials, the focus is to optimize company use of materials to both reduce raw material waste and increase plastics recycling rates; for energy, the focus is to support corporate energy reduction goals by optimizing energy use in laboratories and processes; and for water, the focus is to optimize use of water for production and cleaning while maintaining quality. One of our initial chemical projects was to replace ethidium bromide with less hazardous alternatives—an easy substitution that could be implemented not only at Genentech but also throughout academic laboratories and small companies. We saw rapid adoption of alternatives that were drop-in replacements for our DNAstaining work. Employees tested these replacements during routine laboratory work, generating Genentech-specific data to compare with the supplier’s data. We then shared these results and made suggestions for specific applications and began stocking the alternative stains company-wide. This initiative resulted in an 85% decrease (Fig. 1) in hazardous waste generated from the use of ethidium bromide, resulting in corresponding reductions in waste disposal costs and environmental risk. A growing area in our materials portfolio addresses single-use bioprocessing materials. Single-use materials are playing a larger role in bioprocessing because of their flexibility, reduced infrastructure requirements, and lower water usage due to reduced cleaning requirements. These materials are highly engineered and the biopharma industry should continuously re-evaluate their use for more efficient, innovative ways to incorporate them into processes. We identified an opportunity to rethink the use of single-use materials in our cell culture process and tested new protocols to repurpose single-use media bags for waste collection. Initially, this project was met with skepticism as media bags were regarded strictly as single-use, not surprisingly. However, our project team collected supporting data, engaged employees and obtained leadership endorsement to implement the new protocols. As a result, our pilot plant will see a 91% reduction in the number of media bags used. These and other similar efforts can substantially reduce the costs of production, benefitting all companies, small and large. The vision of green biopharma can only be realized by industry collaboration. To that end, Genentech helped establish the BFG within the GCIPR. The focus group brings together several major pharma companies Figure 1 Reduction in ethidium bromide contaminated hazardous waste after introduction of alternative stains in 2012 at Genentech. 0 200 400 600 800 1,000 1,200 1,400 1,600