Holistic analytical characterization and risk assessment of residual host cell protein impurities in an active pharmaceutical ingredient synthesized by biocatalysts.

Host cell proteins (HCPs) are a significant class of process-related impurities commonly associated with the manufacturing of biopharmaceuticals. However, due to the increased use of crude enzymes as biocatalysts for modern organic synthesis, HCPs can also be introduced as a new class of impurities in chemical drugs. In both cases, residual HCPs need to be adequately controlled to ensure product purity, quality, and patient safety. Although a lot of attentions have been focused on defining a universally acceptable limit for such impurities, the risks associated with residual HCPs on product quality, safety, and efficacy often need to be determined on a case-by-case basis taking into consideration the residual HCP profile in the product, the dose, dosage form, administration route, and so forth. Here we describe the unique challenges for residual HCP control presented by the biocatalytic synthesis of an investigational stimulator of interferon genes protein agonist, MK-1454, which is a cyclic dinucleotide synthesized using Escherichia coli cell lysate overexpressing cyclic GMP-AMP synthase as a biocatalyst. In this study, a holistic characterization of residual protein impurities using a variety of analytical tools including nanoscale liquid chromatography coupled to tandem mass spectrometry, together with in silico immunogenicity prediction of identified proteins, facilitated risk assessment and guided process development to achieve adequate removal of residual protein impurities in MK-1454 active pharmaceutical ingredient.

A kinase-cGAS cascade to synthesize a therapeutic STING activator.

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence1-3. Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents4,5. Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic6,7 (ClinicalTrials.gov study NCT04220866 ). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules.

Quantitation and speciation of residual protein within active pharmaceutical ingredients using image analysis with SDS-PAGE.

Recent advances in biocatalysis and directed enzyme evolution has led to a variety of enzymatically-driven, elegant processes for active pharmaceutical ingredient (API) production. For biocatalytic processes, quantitation of any residual protein within a given API is of great importance to ensure process robustness and quality, pure pharmaceutical products. Typical analytical methods for analyzing residual enzymes within an API, such as enzyme-linked immunosorbent assays (ELISA), colorimetric assays, and liquid chromatographic techniques, are limited for determining only the concentration of known proteins and require harsh solvents with high API levels for analysis. For the first time, total residual protein content in a small molecule API was quantitated using image analysis applied to SDS-PAGE. Herein, a proposed methodology for residual protein detection, quantitation, and size-based speciation is presented, in which an orthogonal technique is offered to traditional analysis methods, such as ELISA. Results indicate that our application of the analytical methodology is able to reliably quantitate both protein standards and the total residual protein present within a final API, with good agreement as compared to traditional ELISA results. Further, speciation of the residual protein within the API provides key information concerning the individual residual proteins present, including their molecular weight, which can lead to improved process development efforts for residual protein rejection and control. This analytical methodology thus offers an alternative tool for easily identifying, quantitating, and speciating residual protein content in the presence of small molecule APIs, with potential for wide applicability across industry for biocatalytic or directed enzyme evolution efforts within process development.

A cost-effective plate-based sample preparation for antibody N-glycan analysis.

During early cell line and process development of therapeutic antibodies, a cost-effective high-throughput approach to characterize the N-linked glycans is highly desired given that a large number of samples need to be analyzed. Using commercially available, low cost 96-well plates, we developed a practical procedure to prepare fluorescently labeled N-linked glycans for both qualitative and quantitative analysis by mass spectrometry (MS) and ultrahigh performance liquid chromatography (UPLC). Antibody samples were continuously denatured, reduced, and deglycosylated in a single 96-well hydrophobic membrane filter plate. Subsequently, released glycans were fluorescently labeled in a collection plate, and cleaned-up using a hydrophilic membrane filter plate. Carried out entirely in ready-to-use 96-well plates with simple buffer systems, this procedure requires less than 90min to finish. We applied the optimized procedure to examine the N-linked glycosylation of trastuzumab and were able to quantify ten major N-linked glycans. The results from different amounts of starting materials (10-200µg) were highly similar and showed the robustness of this procedure. Compared to other methods, this new procedure is simple to implement, economically more affordable, and could be very valuable for early screenings of antibody development.

N-glycosylamine-mediated isotope labeling for mass spectrometry-based quantitative analysis of N-linked glycans.

N-linked glycosylation is a major protein modification involved in many essential cellular functions. Methods capable of quantitative glycan analysis are highly valuable and have been actively pursued. Here we describe a novel N-glycosylamine-based strategy for isotopic labeling of N-linked glycans for quantitative analysis by use of mass spectrometry (MS). This strategy relies on the primary amine group on the reducing end of freshly released N-linked glycans for labeling, and eliminates the need for the harsh labeling reaction conditions and/or tedious cleanup procedures required by existing methods. By using NHS-ester amine chemistry we used this strategy to label N-linked glycans from a monoclonal antibody with commercially available tandem mass tags (TMT). Only duplex experiments can be performed with currently available TMT reagents, because quantification is based on the intensity of intact labeled glycans. Under mild reaction conditions, greater than 95% derivatization was achieved in 30 min and the labeled glycans, when kept at -20 °C, were stable for more than 10 days. By performing glycan release, TMT labeling, and LC-MS analysis continuously in a single volatile aqueous buffer without cleanup steps, we were able to complete the entire analysis in less than 2 h. Quantification was highly accurate and the dynamic range was large. Compared with previously established methods, N-glycosylamine-mediated labeling has the advantages of experimental simplicity, efficient labeling, and preserving glycan integrity.

High-throughput multimodal strong anion exchange purification and N-glycan characterization of endogenous glycoprotein expressed in glycoengineered Pichia pastoris.

The secretory pathway of the yeast Pichia pastoris has been engineered to produce complex human-type N-glycans (Choi et al., Proc Natl Acad Sci USA 100:5022-5027, 2003; Hamilton et al., Science 301:1244-1246, 2003; Hamilton et al., Science 313:1441-1443, 2006). In contrast to the heterogeneous glycans produced on the therapeutic glycoproteins expressed in mammalian cell lines, glycoengineered P. pastoris can be designed to produce a specific, preselected glycoform. In order to achieve glycan uniformity on the target protein, No Open Reading Frame (NORF) yeast cell lines are screened extensively during various stages of glycoengineering. In the absence of the target protein of interest, screening the NORF yeast cell lines for glycoform uniformity becomes a challenge. The common approach so far has been to analyze the total cell glycan pool released from glycoproteins of the NORF yeast cells to predict the N-glycan uniformity. As this does not always accurately predict the N-glycan end product, we describe in this chapter a detailed protocol for a non-affinity-based high-throughput purification of an endogenous glycoprotein. This protein of interest has been introduced during the early stages of glycoengineering process and its N-glycan profile is utilized as a tool for glycoengineering screening.

Elimination of ß-mannose glycan structures in Pichia pastoris.

The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, such as those containing ß-mannose (Man) linkages, can elicit an immune response or bind to Man receptors, thus reducing their efficacy. Recent studies have confirmed that P. pastoris has four genes from the ß-mannosyl transferase (BMT) family and that Bmt2p is responsible for the majority of ß-Man linkages on glycans. While expressing recombinant human erythropoietin (rhEPO) in a developmental glycoengineered strain devoid of BMT2 gene expression, cross-reactivity was observed with an antibody raised against host cell antigens. Treatment of the rhEPO with protein N-glycosidase F eliminated cross-reactivity, indicating that the antigen was associated with the glycan. Thorough analysis of the glycan profile of rhEPO demonstrated the presence of low amounts of α-1,2-mannosidase resistant high-Man glycoforms. In an attempt to eliminate the α-mannosidase resistant glycoforms, we used a systemic approach to genetically knock-out the remaining members of the BMT family culminating in a quadruple bmt2,4,1,3 knock-out strain. Data presented here conclude that the additive elimination of Bmt2p, Bmt3p and Bmt1p activities are required for total abolition of ß-Man-associated glycans and their related antigenicity. Taken together, the elimination of ß-Man containing glycoforms represents an important step forward for the Pichia production platform as a suitable system for the production of therapeutic glycoproteins.

The Vitamin E analog Trolox reduces copper toxicity in the annelid Lumbriculus variegatus but is also toxic on its own.

The ability of the water-soluble Vitamin E analog, Trolox, to prevent the toxic effects of copper exposure on the behavior and neuronal physiology of the freshwater oligochaete Lumbriculus variegatus was examined. Trolox produced a concentration-dependent increase in the 24 h LC(50) for copper exposure, with 100 microM Trolox elevating the LC(50) by almost seven-fold (from 0.36 to 2.43 microM). Copper exposure (0.2 microM) for 24h produced a reduction in the conduction velocity of the medial and lateral giant nerve fibers, which was prevented by 100 microM Trolox. Copper exposure (0.2 microM) for 24h also reduced the effectiveness of substrate vibration in eliciting giant nerve fiber spikes. Trolox prevented this reduction in sensory responsiveness. Trolox (100 microM) partially reversed the copper-induced (0.4 microM) decrease in touch-evoked helical swimming behavior, but had no effect on the copper-induced decrement in touch-evoked body reversal. Copper exposure (0.2 microM) for 24 h reduced the amount of spontaneous locomotion (crawling); however, Trolox did not reverse this effect. However, Trolox exposure alone produced a decrease in the distance crawled that was similar in magnitude to copper exposure. In normal worms, rapid spiking activity of the medial giant nerve fiber produces facilitation in the amplitude of the resulting muscle potentials produced by the longitudinal body wall muscles. Copper exposure had no effect on the amount of muscle potential facilitation, but Trolox exposure (100 microM) produced a significant decrease in facilitation. The results of this study indicate that many of the toxic effects of copper exposure on Lumbriculus are prevented or reduced by the antioxidant Trolox. However, the results of this study also indicate that Trolox has toxic effects on behavior and neuronal physiology. The results presented here document one of the few published reports of the detrimental effects of Vitamin E or its analogs on nervous system function or behavior.