End-to-end collaboration to transform biopharmaceutical development and manufacturing.

An ambitious 10-year collaborative program is described to invent, design, demonstrate, and support commercialization of integrated biopharmaceutical manufacturing technology intended to transform the industry. Our goal is to enable improved control, robustness, and security of supply, dramatically reduced capital and operating cost, flexibility to supply an extremely diverse and changing portfolio of products in the face of uncertainty and changing demand, and faster product development and supply chain velocity, with sustainable raw materials, components, and energy use. The program is organized into workstreams focused on end-to-end control strategy, equipment flexibility, next generation technology, sustainability, and a physical test bed to evaluate and demonstrate the technologies that are developed. The elements of the program are synergistic. For example, process intensification results in cost reduction as well as increased sustainability. Improved robustness leads to less inventory, which improves costs and supply chain velocity. Flexibility allows more products to be consolidated into fewer factories, reduces the need for new facilities, simplifies the acquisition of additional capacity if needed, and reduces changeover time, which improves cost and velocity. The program incorporates both drug substance and drug product manufacturing, but this paper will focus on the drug substance elements of the program.

A common framework for integrated and continuous biomanufacturing.

There is a growing application of integrated and continuous bioprocessing (ICB) for manufacturing recombinant protein therapeutics produced from mammalian cells. At first glance, the newly evolved ICB has created a vast diversity of platforms. A closer inspection reveals convergent evolution: nearly all of the major ICB methods have a common framework that could allow manufacturing across a global ecosystem of manufacturers using simple, yet effective, equipment designs. The framework is capable of supporting the manufacturing of most major biopharmaceutical ICB and legacy processes without major changes in the regulatory license. This article reviews the ICB that are being used, or are soon to be used, in a GMP manufacturing setting for recombinant protein production from mammalian cells. The adaptation of the various ICB modes to the common ICB framework will be discussed, along with the pros and cons of such adaptation. The equipment used in the common framework is generally described. This review is presented in sufficient detail to enable discussions of IBC implementation strategy in biopharmaceutical companies and contract manufacturers, and to provide a road map for vendors equipment design. An example plant built on the common framework will be discussed. The flexibility of the plant is demonstrated with batches as small as 0.5 kg or as large as 500 kg. The yearly output of the plant is as much as 8 tons.

Characterizing the effect of multiple Fc glycan attributes on the effector functions and FcγRIIIa receptor binding activity of an IgG1 antibody.

N-linked Fc glycosylation of IgG1 monoclonal antibody therapeutics can directly influence their mechanism of action by impacting IgG effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Therefore, identification and detailed characterization of Fc glycan critical quality attributes (CQAs) provides important information for process design and control. A two-step approach was used to identify and characterize the Fc glycan CQAs for an IgG1 Mab with effector function. First, single factor experiments were performed to identify glycan critical quality attributes that influence ADCC and CDC activities. Next, a full-factorial design of experiment (DOE) to characterize the possible interactions and relative effect of these three glycan species on ADCC, CDC, and FcγRIIIa binding was employed. Additionally, the DOE data were used to develop models to predict ADCC, CDC, and FcγRIIIa binding of a given configuration of the three glycan species for this IgG1 molecule. The results demonstrate that for ADCC, afuco mono/bi has the largest effect, followed by HM and ß-gal, while FcγRIIIa binding is affected by afuco mono/bi and ß-gal. CDC, in contrast, is affected by ß-gal only. This type of glycan characterization and modeling can provide valuable information for development, manufacturing support and process improvements for IgG products that require effector function for efficacy. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1181-1192, 2016.

Characterization of operating parameters for XMuLV inactivation by low pH treatment.

To ensure the viral safety of protein therapeutics made in mammalian cells, purification processes include dedicated viral clearance steps to remove or inactivate adventitious and endogenous viruses. One such dedicated step is low pH treatment, a robust and effective method commonly used in monoclonal antibody production to inactivate enveloped viruses. To characterize the operating space for low pH viral inactivation, we performed a statistically designed experiment evaluating the effect of pH, temperature, hold duration, acid type, and buffer concentration on inactivation of the retrovirus model, XMuLV. An additional single factor experiment was performed to study the effect of protein concentration. These data were used to generate predictive models of inactivation at each time point studied, which can be used to identify conditions for robust and effective XMuLV inactivation. At pH 3.6, XMuLV inactivation was rapid, robust, and relatively unaffected by the other factors studied, providing support for this as a generic viral inactivation condition for products that can tolerate this low pH. At pH 3.7 and 3.8, other factors besides pH affected XMuLV inactivation. By understanding the impact of each factor on inactivation, the factors can be manipulated within the operating space to ensure effective inactivation while achieving desired product quality goals.

Differential binding of heavy chain variable domain 3 antigen binding fragments to protein A chromatography resins.

This work examines the binding of 15 different VH3 IgGs and their corresponding F(ab')2 fragments to two different protein A chromatography resins: MabSelect(®), which utilizes a recombinant protein A ligand, and MabSelect SuRe(®) (SuRe), which utilizes a tetrameric Z domain ligand. The results show that VH3 F(ab')2 fragments can exhibit a variety of binding behaviours for the two resins. Contrary to previously published data, a subset of these molecules show strong interaction with the Z domain of SuRe(®). Furthermore, the results show that sequence variability of residue 57 in the VH3 heavy chain CDR2 domain correlates with binding behaviour on MabSelect(®) and SuRe(®). Site-directed mutagenesis of this residue confers gain or loss of VH3 F(ab')2 binding to these resins in 3 mAbs, demonstrating that it plays a key role in both recombinant protein A and Z domain interaction. A fourth mAb with a longer CDR2 loop was not affected by mutation of residue 57, indicating that CDR2 domain length may alter the binding interface and lead to the involvement of other residues in protein A binding.

The criticality of high-resolution N-linked carbohydrate assays and detailed characterization of antibody effector function in the context of biosimilar development.

Accurate measurement and functional characterization of antibody Fc domain N-linked glycans is critical to successful biosimilar development. Here, we describe the application of methods to accurately quantify and characterize the N-linked glycans of 2 IgG1 biosimilars with effector function activity, and show the potential pitfalls of using assays with insufficient resolution. Accurate glycan assessment was combined with glycan enrichment using lectin chromatography or production with glycosylation inhibitors to produce enriched pools of key glycan species for subsequent assessment in cell-based antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity effector function assays. This work highlights the challenges of developing high-quality biosimilar candidates and the need for modern biotechnology capabilities. These results show that high-quality analytics, combined with sensitive cell-based assays to study in vivo mechanisms of action, is an essential part of biosimilar development.

Clearance of the rodent retrovirus, XMuLV, by protein A chromatography.

Protein A chromatography is the most common unit operation used in the manufacture of therapeutic monoclonal antibodies (mAbs) due to its high affinity and specificity for the IgG Fc domain. However, protein A chromatography is often not effective for viral clearance. Typical log reduction values (LRV) for the model retrovirus XMuLV range between 1 and 4 logs, while effective steps such as viral filtration can achieve 5-7 logs of clearance. XMuLV LRVs obtained on protein A are reproducible for a given mAb, but can vary widely for different mAbs, even with the same operating conditions. In order to understand the mechanism of XMuLV clearance on protein A, we have investigated its partitioning on Mabselect SuRe protein A resin and explored how the virus interacts with resin, product, and impurities. The results show that XMuLV has some interaction with the resin backbone and ligand, but also appears to bind to and coelute with the mAb. The interaction with product was further examined by evaluating the effect of feed conditions, loading, and different washes on XMuLV partitioning on the column. Understanding the mechanism of XMuLV removal on a protein A, resin provides insight into the variability and low viral clearance of this step and suggests ways in which the removal of virus by this step can be improved.

Use of MMV as a Single Worst-Case Model Virus in Viral Filter Validation Studies.

Typical platform processes for biopharmaceutical products derived from animal cell lines include a parvovirus filtration unit operation to provide viral safety assurance of the drug product. The industry has adopted this platform unit operation and gained a wider understanding of its performance attributes, leading to the possibility of streamlined approaches to virus clearance validation. Here, the concept of virus validation on a parvovirus-grade filter with a single worst-case model virus is presented. Several lines of evidence, including published literature and Amgen's own data, support the use of a parvovirus, such as mouse minute virus (MMV), as a worst-case model virus to assess virus removal by parvovirus filters. The evidence presented includes a discussion of the design and manufacture of virus filters with a size exclusion mechanism for removal. Next, the characteristics of different model viruses are compared and a risk assessment on the selection of the relevant model viruses for clearance studies is presented. Finally, a comprehensive summary of literature and Amgen data is provided, comparing the clearance of larger viruses against MMV. Together, this analysis provides a strong scientific rationale for the use of a single, worst-case model virus for assessing virus removal by parvovirus filters, which will ultimately allow for more efficient and streamlined viral clearance study designs. LAY ABSTRACT: Demonstrating the virus clearance capability of a purification process is an important aspect of biopharmaceutical process development. A key component of the viral safety of the process is the inclusion of a parvovirus-grade filter as an effective and robust virus removal step. Traditional methodologies for viral clearance studies have been based on a conservative, data-intensive approach, but recent trends in the field of virus clearance and process development show evolution towards streamlined and more efficient study designs that are based on understanding the mechanism of viral clearance by downstream unit operations. The publication of scientific datasets and awareness of the underlying mechanisms involved with these unit operations have fueled this trend. Here, the concept of virus validation on a parvovirus-grade filter using a parvovirus as single, worst-case model virus is presented. Multiple lines of evidence are provided to support this proposal, including a review of published literature and Amgen historical data. The adoption of this approach provides benefits in terms of cost savings for executing viral clearance studies, but it also simplifies the necessary dataset and focuses on only supplying value-added information to demonstrate the viral safety of the process.

Hydroxocobalamin association during cell culture results in pink therapeutic proteins.

Process control of protein therapeutic manufacturing is central to ensuring the product is both safe and efficacious for patients. In this work, we investigate the cause of pink color variability in development lots of monoclonal antibody (mAb) and Fc-fusion proteins. Results show pink-colored product generated during manufacturing is due to association of hydroxocobalamin (OH-Cbl), a form of vitamin B12. OH-Cbl is not part of the product manufacturing process; however we found cyanocobalamin (CN-Cbl) in cell culture media converts to OH-Cbl in the presence of light. OH-Cbl can be released from mAb and Fc-fusion proteins by conversion with potassium cyanide to CN-Cbl, which does not bind. By exploiting the differential binding of CN-Cbl and OH-Cbl, we developed a rapid and specific assay to accurately measure B12 levels in purified protein. Analysis of multiple products and lots using this technique gives insight into color variability during manufacturing.

Using high throughput screening to define virus clearance by chromatography resins.

High throughput screening (HTS) of chromatography resins can accelerate downstream process development by rapidly providing information on product and impurity partitioning over a wide range of experimental conditions. In addition to the removal of typical product and process-related impurities, chromatography steps are also used to remove potential adventitious viral contaminants and non-infectious retrovirus-like particles expressed by rodent cell lines used for production. This article evaluates the feasibility of using HTS in a 96-well batch-binding format to study removal of the model retrovirus xenotropic murine leukemia virus (xMuLV) from product streams. Two resins were examined: the anion exchange resin Q Sepharose Fast Flow™ (QSFF) and Capto adhere™, a mixed mode resin. QSFF batch-binding HTS data was generated using two mAbs at various pHs, NaCl concentrations, and levels of impurities. Comparison of HTS data to that generated using the column format showed good agreement with respect to virus retentation at different pHs, NaCl concentrations and impurity levels. Results indicate that NaCl concentration and impurity level, but not pH, are key parameters that can impact xMuLV binding to both resins. Binding of xMuLV to Capto adhere appeared to tolerate higher levels of NaCl and impurity than QSFF, and showed some product-specific impact on binding that was not observed with QSFF. Overall, the results demonstrate that the 96-well batch-binding HTS technique can be an effective tool for rapidly defining conditions for robust virus clearance on chromatographic resins.

Cation exchange chromatography provides effective retrovirus clearance for antibody purification processes.

One measure taken to ensure safety of biotherapeutics produced in mammalian cells is to demonstrate the clearance of potential viral contaminants by downstream purification processes. This paper provides evidence that cation exchange chromatography (CEX), a widely used polishing step for monoclonal antibody (mAb) production, can effectively and reproducibly remove xMuLV, a retrovirus used as a model of non-infectious retrovirus-like particles found in Chinese hamster ovary cells. The dominant mechanism for xMuLV clearance by the strong cation exchanger, Fractogel SO 3⁻, is by retention of the virus via adsorption instead of inactivation. Experimental data defining the design space for effective xMuLV removal by Fractogel SO 3⁻ with respect to operational pH, elution ionic strength, loading, and load/equilibration buffer ionic strength are provided. Additionally, xMuLV is able to bind to other CEX resins, such as Fractogel COO⁻ and SP Sepharose Fast Flow, suggesting that this phenomenon is not restricted to one type of CEX resin. Taken together, the data indicate that CEX chromatography can be a robust and reproducible removal step for the model retrovirus xMuLV.

Photochemical degradation of citrate buffers leads to covalent acetonation of recombinant protein therapeutics.

Novel acetone and aldimine covalent adducts were identified on the N-termini and lysine side chains of recombinant monoclonal antibodies. Photochemical degradation of citrate buffers, in the presence of trace levels of iron, is demonstrated as the source of these modifications. The link between degradation of citrate and the observed protein modifications was conclusively established by tracking the citrate decomposition products and protein adducts resulting from photochemical degradation of isotope labeled (13)C citrate by mass spectrometry. The structure of the acetone modification was determined by nuclear magnetic resonance (NMR) spectroscopy on modified-free glycine and found to correspond to acetone linked to the N-terminus of the amino acid through a methyl carbon. Results from mass spectrometric fragmentation of glycine modified with an acetone adduct derived from (13)C labeled citrate indicated that the three central carbons of citrate are incorporated onto protein amines in the presence of iron and light. While citrate is known to stoichiometrically decompose to acetone and CO(2) through various intermediates in photochemical systems, it has never been shown to be a causative agent in protein carbonylation. Our results point to a previously unknown source for the generation of reactive carbonyl species. This work also highlights the potential deleterious impact of trace metals on recombinant protein therapeutics formulated in citrate buffers.

Drosophila lacking the Cdk5 activator, p35, display defective axon guidance, age-dependent behavioral deficits and reduced lifespan.

The cyclin-dependent kinase Cdk5 has attracted a great deal of attention both because of its roles in cell migration and axon patterning, and the extensive data implicating it in adult-onset neurodegeneration in mammals. Both the kinase activity and the biological effects of Cdk5 are absolutely dependent on association with an activating subunit, called p35. We show here that Drosophila lacking the Cdk5 activator, D-p35, display a wide range of defects in embryonic axon patterning. We further show that, while viable and fertile, p35 mutant adults display progressive, age-dependent loss of motor function and have a significantly shortened lifespan.