Cynthia A. Challener is a contributing editor to Pharmaceutical Technology.

Cynthia A. Challener

	Residual undesired proteins-host-cell proteins (HCPs) and high-molecular-weight (HMW) and low-molecular-weight (LMW) species-have the potential to affect the safety and efficacy of biopharmaceuticals. As a result, the levels of these protein impurities in biologic drug substances and drug products must be controlled and are typically considered as critical quality attributes. The properties of the different contaminants vary significantly. In addition, the identification and quantification of some of these residual proteins, particularly in the presence of large concentrations of the product protein, can be challenging. Use of multiple orthogonal analytical methods and purification techniques is often required. Advances in mass spectrometry are, however, enabling more detailed analyses and thus improved monitoring of residual proteins during downstream processing.

	The most common protein-based impurities that downstream processes are developed to control include high- and low-molecular-weight species, charge-variants, and host-cell proteins.

	HMW species include dimers, trimers, tetramers, etc., formed of monomers that can be either covalently or non-covalently linked, according to John Moscariello, vice-president of process development for CMC Biologics. “Often, these large protein impurities consist of misfolded monomers in which surfaces of the monomer are exposed that typically would not be in the monomeric form. They are believed to primarily impact safety, but also can impact efficacy,” he says. He does note, however, that there are data suggesting that some aggregated species are well tolerated. Even so, regulatory agencies expect biopharmaceutical manufacturers to control HMW species through specifications for the drug substance, drug product, and end-of-shelf life.

	LMW species, which include clipped species and half molecules for compounds that are intended to be dimeric, such as monoclonal antibodies and bispecific antibodies, must also be controlled during downstream processing. These molecules can impact both safety due to the potential to cause immunogenicity responses and efficacy as the result of missing structural features, such as complementarity determining regions, which play a role in determining the antigen-binding specificities of antibodies and T-cell receptors, according to Moscariello.

	HCPs are generated during the production of recombinant proteins. The host cells used for fermentation/cell culture produce HCPs necessary for the cells to function (growth, gene transcription, protein synthesis, etc.) and during cell death. They must also be controlled during downstream processing because they have the potential to elicit an immune response in patients due to the use of non-human cell lines (typically Chinese hamster ovary or CHO cells) for production. Some HCPs can also affect drug product stability. “Regulatory agencies expect robust control of these species through drug substance release testing,” Moscariello observes.

	The properties of the different types of residual protein contaminants in biologic drugs vary significantly, leading to the need to use different analytical methods to detect and monitor each. The presence of HMW species, for instance, is frequently determined using size-exclusion chromatography (SEC) coupled with high-pressure liquid chromatography (HPLC) or ultra-high performance liquid chromatography (UHPLC), while LMW species are detected using a capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) (reduced and non-reduced) technique. HCPs are typically measured using enzyme-linked immunosorbent assays (ELISA).

	“SEC-HPLC (or UHPLC) is a very robust assay and typically has very good resolution for HMW species, but it is often difficult to resolve LMW species,” Moscariello says. As a result, CE-SDS is used for the detection of LMW protein impurities. The SDS denatures the protein, and if needed a reducing agent can be added to break any disulfide bonds. As in SDS-PAGE (polyacrylamide gel electrophoresis), the LMWs are separated based on size. For monoclonal antibodies, when running a reduced CE-SDS, typically highly resolved intact heavy and light chains, as well as any clipped species or non-reducible HMW species (i.e., covalently linked compounds) can be observed, according to Moscariello. To detect half-antibodies, however, non-reduced CE-SDS is frequently used because they are difficult to detect when using a reducing agent.

	Although ELISA is the most common technique for monitoring HCPs, the selectivity of this method is dependent on the antibodies used in the ELISA kit, and consequently some HCPs can potentially be missed, according to Peter Levison, senior marketing director, downstream processing with Pall Life Sciences. HCP ELISAs employ a polyclonal mixture of anti-host cell proteins, but often do not provide complete coverage for all of the HCPs expressed during a specific process. This situation is particularly true for commercial kits. “While these kits allow for off-the-shelf detection of a wide range of HCPs for various cell lines, including CHO, they likely have significant gaps in HCPs for each specific process. As a result, ELISAs are known to show significant variability and it is difficult to get accurate values,” Moscariello comments. On the other hand, he notes that the development of product-specific HCP assays with high coverage is costly and time consuming.

	Most companies are exploring more advanced techniques for the detection of HCPs and other protein impurities that are designed to overcome the limitations of existing methodologies. In particular, Moscariello notes significant investment in process analytical technology (PAT) tools for online or at-line monitoring of HMWs and other contaminants. For instance, he points to the use by Amgen of multi-angle light scattering (MALS) to monitor protein aggregates at the effluent of a cation-exchange column.

	Several advances in detection techniques for HCPs are also being made. Use of isotope tags for relative and absolute quantification (iTRAQ) HPLC technology can enable better detection of host-cell proteins, and when coupled with comprehensive genome sequences, can enable better identification of HCPs using established mass spectrometry (MS) databases, according to Levison. Data obtained from techniques such as surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF) MS in combination with ELISA results are also allowing semi-quantification of total HCPs.

	The first step in the purification of bioprocess fluids taken from bioreactors is clarification. In this step, the biologic-drug substance is separated from cell debris and turbidity is removed, leaving a clear harvested cell-culture fluid. This step is typically achieved via depth filtration or centrifugation and does also lead to the reduction of undesired protein impurities. Pall Life Sciences introduced the Cadence Acoustic Separator technology in April 2016-an alternative clarification method for cell-culture bioprocess fluids, including those from continuous perfusion processes-based on acoustic wave separation technology licensed in 2015 from FloDesign Sonics (FDS).

	Following clarification, orthogonal chromatographic steps during protein purification are generally used to further reduce HCP, HMW, and other contaminants to acceptable levels, according to Levinson. “The most powerful step to remove HCPs is affinity chromatography,” asserts Moscariello. There are issues with this method, however. While almost universally adopted for monoclonal antibodies, this method is often not applicable for non-fragment-crystallizable (Fc)-region-containing products. There are significant product-HCP interactions with antibodies. It is common to try to disrupt these interactions with harsh washes, particularly on affinity resins, which requires that they be salt-tolerant. Levison adds that the introduction of new mixed-mode modalities has provided additional selectivities that are providing improved HCP clearance.

	There is also a lot of work being done with precipitating and flocculating agents, according to Moscariello. He notes that caprylic acid has been shown to significantly remove HCPs from product streams without significant loss of product.

	Advances in detection technologies are having an impact on the capability of downstream processing to remove undesired protein impurities, according to Moscariello “The ability to identify specific HCPs using state-of-the-art MS techniques has allowed downstream development scientists to assess the charge, size, and hydrophobicity of these HCPs and exploit their differences from the product of interest and develop more robust processes,” Moscariello adds.

	“Improvements in analytics and genomic/proteomic analyses may well provide better awareness of the presence of contaminants and therefore facilitate their clearance,” Levinson concludes.

	When referring to this article, please cite it as C. Challener, "Protein Impurities Pose Challenges,” 

Articles

Multiple methods are required for detecting and removing protein impurities.

Protein Impurities Pose Challenges

Continuous flow manufacturing for small-molecule drug synthesis received a vote of confidence in April 2016 when FDA approved a change from batch to continuous processing for Janssen Products’ Prezista (darunavir), a drug for the treatment of HIV-1 infection. This decision is the first such approval for a change from batch to continuous manufacturing, and the second for a continuous process; Vertex received approval in July 2015 for the continuous production of its cystic fibrosis drug Orkambi (lumacaftor/ivacaftor).

	In a blog posted on April 12, 2016, Lawrence Yu, FDA’s deputy director of the Office of Pharmaceutical Quality in the Center for Drug Evaluation and Research, expounded on the benefits of continuous manufacturing: greater reliability, safety, efficiency, responsiveness/flexibility, and reduced costs (1).

	He stated, “Although it is not easy for drug manufacturers to transition from batch to continuous manufacturing, there are significant rewards. FDA encourages others in the pharmaceutical industry to consider similar efforts.”

	Pharmaceutical companies are certainly interested as well, as indicated by the numerous reports of API synthetic methods designed to be operated under continuous flow conditions. A summary of selected processes is presented in the following.


	Researchers in China report a continuous-flow process for the preparation of 

-nitroaniline to afford a diazonium chloride intermediate, which is then subjected to azo coupling with sodium thiomethoxide to give 1-(methylthio)-2-(3-nitrophenyl)diazene, followed by dediazonization to provide 

-nitrothioanisole in high yield (2). A flow process was developed to minimize accumulation of the energetic intermediate diazonium salt and enable large-scale production.

	The same scientists also developed a continuous flow process for the selective nitration of 1-methyl-4-(methylsulfonyl)benzene in 98% yield using 80 weight percent (wt%) sulfuric acid (3). The short residence time of 5 s led to a significant reduction in byproduct formation. Notably, the spent acid could be reused multiple times after concentration under a vacuum.

	Swedish researchers established a protocol for the transcarbamylation/transesterification of sulfonylcarbamates with alcohols via microwave heating under continuous-flow conditions (4). Using transparent borosilicate tube reactors, the scientists prepared two series of O-alkylsulfonylcarbamates. They also applied the methodology to the synthesis of three angiotensin II type 2 receptor ligands.

	Scientists at the University of Graz (Austria) and Noramco report the continuous flow production of hydrocodone from thebaine via selective olefin reduction to form 8,14-dihydrothebaine followed by hydrolysis (5). Selective transfer hydrogenation using hydrazine hydrate (N

 is not possible under batch conditions due to the high explosion potential of the reaction mixture. The reported continuous high-temperature/high-pressure method is efficient, safe, and scalable. The process involves four consecutive feeds of liquid N

O with separate residence times for completion of the reaction in 1 h.

	Researchers from The Netherlands developed an intensified, solvent-free continuous process for the conversion of alcohols to chlorides using hydrogen chloride gas (6). A separate dry zone was used to deliver the hydrogen chloride gas and prevent corrosion. Less than half the equivalents of HCl are needed due to the use of the gas instead of hydrochloric acid, and only water is generated as a byproduct. The reaction is typically complete within 20 min.

	Scientists from Eli Lilly and Company and Vanderbilt University reported the conversion of a batch-based stereoselective aza-Henry reaction between an arylnitromethane and Boc-protected aryl aldimine using a homogeneous Brønsted acid-base catalyst to an automated intermittent-flow process with product crystallization and slow reagent addition (7). The organocatalytic enantioselective reaction included recycle of the catalyst, resulting in increased process intensity maintaining a high catalyst concentration in the reactor. The researchers performed a continuous campaign with the automated system and obtained high throughput and high selectivity while minimizing catalyst usage.

	Researchers from Eli Lilly have also achieved a continuous Grignard reaction using a continuous stirred tank reactor (CSTR) (8). The process was developed after mapping the operation space using knowledge about CSTR process parameters, the reaction kinetics, magnesium (Mg) sequestration efficiency, equipment characterization, and the impact of process disturbances on steady-state conditions. A reaction kinetics model was also developed, and computational fluid dynamics (CFD) models were applied to the equipment design. This approach enabled the development of a highly robust and safe commercial-scale process.

	Researchers at AztraZeneca, in combination with various European university groups, have focused on the development of continuous Buchwald-Hartwig reactions. In one report, the scientists used the highly active palladium N-heterocyclic carbene (NHC) precatalyst [Pd(IPr*)(cin)Cl] 4 (IPr* = 1,3-bis(2,6-bis(diphenylmethyl)-4-methylphenyl)imidazol-2-ylidene; cin = η3-cinnamyl), a universal approach for continuous workup and purification, and a methodology for catalyst recycling and reuse (9). Before implementing the flow reaction, the catalyst’s stability in water was investigated and an effective workup methodology was developed that allows separation of the Buchwald-Hartwig product into the aqueous stream as a salt while retaining the aryl bromide starting material in the organic stream with the catalyst.

	The flow reaction was performed in a microreactor at the gram scale using continuous workup conditions involving acidic extraction of the product and on-stream recycling of the catalyst in the organic phase (10). The custom-designed rector for running heterogeneous reactions in flow combines the flexibility of CSTRs with the smooth operation, low residence time distribution and excellent heat transfer capability of a conventional flow reactor, according to the researchers.

Low-temperature batch to continuous processes


	The conversion of low-temperature batch reactions to continuous processes is attractive because in many cases higher temperatures can be used to achieve equal or better results. For example, scientists from Eisai report the continuous production of eribulin mesylate via the diisobutylaluminum hydride reduction of an ester followed by n-butyllithium-mediated coupling of the resultant aldehyde to a sulfone fragment to afford a sulfone-diol as a key intermediate, all without the need for cryogenic conditions (11). The reduction was run at -50 °C instead of -70 °C, yet a better selectivity and yield were obtained, while the butyllithium-mediated coupling when run under flow conditions at 10 °C proceeded with a better conversion rate than the batch reaction when performed at -70 °C.

	Meanwhile, scientists at Bayer Technology Services report the running of a continuous process for the lithiation of fluoroaromatics in a microreactor demonstration unit (12). A higher temperature was possible in this case, too, leading to higher space-time yields compared to those observed for the batch reaction. Temperature control was found to be critical, though; optimization was necessary to identify the most appropriate reaction conditions, hardware, and reactant purification methods to achieve minimal salt precipitation and prevent runaway conditions.

	Investigation of continuous processing has not been limited to chemical syntheses. There has also been significant interest in developing continuous biocatalytic reactions, which are more complex and challenging to implement. German researchers reported the integration of microfluidic reactor technology with solid-liquid biocatalytic reactions (13). As a case study, they reported the biocatalytic conversion of styrene to (S)-styrene oxide using catalytic biofilms in a microreactor.

., Article ASAP, Web publication, March 24, 2016.
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. 20 (2), 480-486 (2016).
	13. R. Karande, A. Schmid, and K. Buehler, 

Research efforts around the world are demonstrating that the application of flow chemistry for small-molecule API synthesis.

Flow Chemistry Discoveries Advance Continuous Processing

The majority of small-molecule drugs in development today contain chiral centers, and according to Global Industry Analysts (GIA) nearly 95% of drugs will be chiral by 2020 (1). GIA estimates that the global chiral technology market, which is dominated by pharmaceutical applications (followed by agrochemicals and flavors and fragrances), will reach $5.1 billion by 2017.

	Because FDA requires manufacturers to investigate the properties (physicochemical, pharmacokinetic, etc.) of all enantiomers/diastereomers of chiral drugs to determine their individual safety and efficacy, the development of efficient chiral synthesis technologies remains a primary target for many academic and industrial researchers. Several transformations with significant potential for use in API manufacture have been published in the past year. A few of them are highlighted in the following.


	Researchers in the Doyle lab at Princeton University in New Jersey have developed a direct cross-coupling reaction to produce 1,2-dihydropyridines, versatile building blocks that are highly useful for API synthesis (2). The reaction involves the enantioselective Ni-catalyzed cross coupling of arylzinc reagents with pyridinium ions formed 

	The use of pyridine is of particular interest, according to Abigail Doyle, an associate professor of chemistry at Princeton and corresponding author of the article. “A highlight of the method is being able to use pyridine as a substrate because it’s inexpensive and abundant and has rarely been used in transition metal and asymmetric catalysis,” she said (3). Pyridine typically binds to nickel catalysts, inhibiting further catalytic activity. This issue was avoided by adding a slight excess of iso-butylchloroformate, an activating agent that reacts with pyridine to form an intermediate species that does not bind to the catalyst.

	The generated, enantioenriched 2-aryl-1,2-dihydropyridine products can be elaborated to numerous piperidine derivatives with little or no loss in enantiomeric excess. Nine different elaborations commonly used by medicinal chemists for drug development were performed to demonstrate their utility (3).

Radical strategy for enantioselective carbon-carbon bond formation


	Most enantioselective reactions proceed via polar reactivity, with few radical reactions providing high selectivities. Radical mechanisms are attractive for forming quaternary carbon centers, however, because radical intermediates are not significantly affected by steric factors, as is the case with cationic and anionic intermediates. Researchers at the Institute of Chemical Research of Catalonia (ICIQ) in Spain have recently reported a method that harnesses the potential of radicals for enantioselective synthesis of quaternary carbon stereocenters (4). Paolo Melchiorre and his group at ICIQ used a combination of photoredox and asymmetric organocatalysis to mediate enantioselective radical conjugate additions to β,β-disubstituted cyclic enones.

	A chiral organocatalyst containing a redox-active carbazole moiety was designed to generate iminium ions that are then stereoselectively trapped via an electron-relay mechanism by photochemically generated carbon-centered radicals. The reaction proceeds under mild reaction conditions with high enantioselectivity and without the need for transition-metal catalysts, making it a sustainable process (5). “Combining the potential of metal-free enantioselective organocatalysis and visible light photocatalysis can significantly advance the development of a more responsible and sustainable stereoselective chemistry while strengthening the chemistry toolbox to better face the challenges of modern organic chemistry,” noted Melchiorre. He added that “This catalyst may provide further opportunities to stereoselectively generate quaternary stereocenters that cannot be forged by using traditional polar reactivity.”

-symmetrical, atropoisomeric functionalized biaryls


	Organocatalysis was also employed by scientists at Rice University in Texas to achieve the direct arylation of aromatic C(sp

)-H bonds in phenols and naphthols for the preparation of 1,1Ê¹-linked functionalized biaryls (6). Not only have the non-C

-symmetrical, atropoisomeric products not been previously synthesized, they are formed with complete regioselectivity, and the reaction is readily scalable and does not require an external oxidant. Results of density-functional theory calculations suggest that the reactions proceed via an asynchronous [3,3]-sigmatropic rearrangement of an 

	The biaryls are synthesized from readily available compounds, including quinone monoacetals and phenols/ naphthols. “Using these building blocks, we made 41 different chiral biaryl compounds in a relatively short time,” said corresponding author László Kürti (7). He added that the functionalized chiral biaryls are really versatile compounds. “You can use them outright as organocatalysts or complex them with transition metals to make new transition-metal catalysts. So the possibilities are unlimited. Moreover, these compounds can be used as building blocks en route to natural products with biaryl substructures in them.”


	Researchers at the University of Sydney in Australia have focused on the development of switchable phosphine ligands for transition metals whose chirality is modulated through an external trigger signal. These molecular motors make it possible to change the enantioselectivity in a catalytic process without the need to synthesize new catalysts. In the report, a photoswitchable chiral bisphosphine was prepared based on a unidirectional light-driven molecular motor that can be used to invert the stereoselectivity of a palladium-catalyzed desymmetrization reaction (8). As a result, each enantiomer can be formed using just one ligand.

	The bisphosphine ligand undergoes photochemical and thermal isomerization during unidirectional rotation around the motor central double bond, allowing for control of both the helicity of the ligand and the distance between the two phosphines. Notably, light-induced isomerizations in the ligand are endothermic, while heat-induced isomerizations are exothermic (9). The researchers report obtaining a nearly racemic mixture to very high enantiomeric ratios for each isomer (93:7 and 6:94) for the Pd-catalyzed desymmetrization reaction (8).

Understanding how chiral molecules behave


	Scientists at Newcastle University in England and the Swiss Federal Laboratories for Materials Science and Technology are learning about mechanisms of chirality through investigation of biomineralization, a process by which inorganic materials are formed with chiral structures following contact with proteins and polysaccharides, which act as modifiers during nucleation and crystal growth (10). The chirality of the biomolecules is transferred to the mineral structure; understanding how this process occurs can provide insights into the design of organic chiral transformations.

	In this report, Werner Hofer and his colleagues used scanning tunneling microscopy, photoelectron diffraction, and density functional theory to investigate how the chiral compound ‘buckybowl’ hemibuckminsterfullerene arranges copper surface atoms. “By understanding this process, we can now force materials to behave in a certain way, using biological plans to create the shapes and structures that we want. This has huge potential in the fields of materials design and drug synthesis,” Hofer said (11). In particular, the knowledge gained could facilitate the development of more efficient and effective enantioselective heterogeneous catalysts.
	Now that the proof of concept has been demonstrated, the challenge, according to the researchers, is to identify the right biomolecules that create the specific metal surface morphologies that are suitable for specific catalytic reactions (11).


	1. Global Industry Analysts, “Global Chiral Technology Market to Reach US$5.1 Billion by 2017, According to New Report by Global Industry Analysts, Inc.,” Press Release, April 4, 2012.
	2. J.P. Lutz, et al., 

. (2016). Advance article. DOI: 10.1039/C6SC00702C.
	3. Princeton University, “New Reaction Turns Feedstock Chemical Into Versatile, ChiraBuilding Block,” Press Release (Princeton, NJ, March 23, 2016).
	4. J.J. Murphy, et al., 

, 532 (7598), 218-222 (2016). DOI:10.1038/nature17438.
	5. Institute of Chemical Research of Catalonia, “Radical Route For The Synthesis Of Chiral Molecules,” Press Release (Tarragona, Spain, April 14, 2016).
	6. H. Gao, et al., Angewandte Chemie International Edition, 55 (2), 566-571 (2016).
	7. Rice University, “Chemical Design Made Easier,” Press Release (Houston, TX, Nov. 24, 2015).
	8. D. Zhao et al., 

., 6, 6652 (2015).
	9. The University of Sydney, “Molecular Motors For Chiral Catalysis,” Press Release (Sydney, Australia, October 2015).
	10. W. Xiao et al., 

, 8, 326-330 (2016).
	11. Newcastle University, “Nature’s Mirror-the Code for Chirality,” Press Release, (Newcastle upon Tyne, England, Feb. 8, 2016).

Pharmaceutical Technology
	Vol. 40, No. 7
	Pages: 28–29

When referring to this article, please cite as C. Challener, "Expanding the Chiral Toolbox," 

Recent chiral advances demonstrate promise for API synthesis.

Expanding the Chiral Toolbox

Biologic drug substances must meet numerous specifications based on critical quality attributes that impact the safety and efficacy of the active ingredient. These important properties must be consistent from batch to batch and within batches if, after final filtration, the API is collected in multiple containers rather than one large vessel. Biologic API uniformity can, however, be influenced by many aspects of the production process, including the quality and variability of the raw materials. Effective harvesting and purification processes play an essential role in ensuring that biopharmaceutical manufacturing processes provide biologic drug substances with uniform and consistent properties.


	When formulating biopharmaceutical drug products, it is essential that the biologic API have consistent product properties, regardless of the production batch or container in which it was collected following final filtration. Variation in the concentration of product- and/or process-related impurities is of paramount importance as it can affect the safety and/or efficacy of the product, according to Brian G. Turner, senior director of manufacturing sciences and technology with the Specialty Care Operations group within Sanofi. “Product-related impurities such as aggregates can be difficult to control at low levels, and certain process-related impurities can be challenging to reduce to acceptable levels if their physicochemical properties are similar to the desired product,” he notes.


	API consistency begins upstream with cell line selection, according to Tim Hill, director of upstream process development for Fujifilm Diosynth Biotechnologies USA. “If a mammalian cell line is not entirely clonal or is sensitive to small changes in processing controls such as pH, temperature, gassing, and feeds, we typically see wide swings in cell growth, metabolites, and production profiles and impacts on product quality attributes such as aggregates, charged variants, and glycans,” he explains.

	The use of undefined media components such as soy or yeast hydrolysates are particularly problematic for API product quality consistency. Lot-to-lot variability of the undefined components impacts cell cultures more than fermentation processes and typically requires raw-material use testing prior to acceptance into manufacturing, according to Hill. Fortunately, he notes that several media vendors have developed very good chemically defined (CD) alternatives to traditional growth media and feeds for common cell culture applications.

	Isolating a stable cell line with robust growth and protein expression capability is still the major challenge in cell culture, Hill adds. “Because of the increased interest in the manufacture of biosimilar monoclonal antibodies, cell clone selection with a close eye on product quality is even more important to ensure that undesirable qualities such as aggregates, half-antibodies, and high mannose-5 glycan derivatives are screened out of the process,” he says.


	While some products may benefit from maintaining tight control of harvesting pH and temperature, Turner finds that the impact of harvesting on product purity is often minimal for secreted proteins, but can be significant for proteins expressed intracellularly, such as insulin expressed as insoluble inclusion bodies in 

	The harvesting process must, however, provide a relatively uniform in-process material to the start of downstream operations with each production batch. To achieve this goal, according to Hill, requires consistency in processing times to avoid over exposure to proteases from lysed cells and unfavorable physiochemical conditions for the protein. Tighter control of the harvest tank environment to minimize degradation of certain products may also have a positive impact on API purity, according to Turner. “When raw materials or process controls vary too much, the consistency of the harvested material can change, which can impact downstream process performance,” he says. “Clearance of potential contaminants including viruses and chemical process impurities during downstream processing can often be impacted by upstream variability, leading to failure in release specifications related to process safety measures,” Hill continues.

	Some advances in technology are helping biologic API manufacturers improve the harvesting process with respect to achieving more consistent harvest cell-culture fluids (HCCFs). For instance, Turner notes that the use of flocculants to improve flows (throughput) in centrifugation can improve the efficiency and increase the capacity of post-centrifuge depth filters. In addition, charged depth filters can reduce process-related impurities such as DNA and basic proteins. Other technology advances are leading to improved efficiencies while also ensuring product consistency. “High-capacity depth filters that can process high-density cultures without centrifugation provide increased efficiencies, while process intensification (e.g., concentrated fed-batch culture) using alternating tangential flow (ATF) filtration allows for the continuous harvest of clarified supernatant,” Turner notes.

	For Hill, single-use tubular bowl centrifuges represent a key technology development for harvesting. These centrifuges collect cells in a gentle manner to minimize cell breakage. “Because this type of centrifuge enables harvesting at high-cell viability, it allows for lower protease, host-cell protein (HCP), and DNA content in the clarified harvest, and thus better capture column performance downstream,” he observes.

	With respect to future developments in harvesting technology, Turner would welcome advances in depth filter construction that would enable the effective processing of high-density cultures. The key remaining challenge, according to Hill, relates to the need to reduce impurity loads associated with in-process streams; lower impurity concentrations would enable the reduction in size of purification columns. Fujifilm Diosynth Biotechnologies is, in fact, working with 3M to evaluate the capability of 3M’s new Emphase AEX Hybrid Purifier to decrease DNA and HCP concentrations in cell-culture harvests. “This additional filtration technology appears to significantly reduce DNA loads onto Protein A capture columns, thus enabling the capture columns to more effectively separate host-cell proteins from biologic APIs,” comments Hill.


	“A well-designed purification process should deliver a consistent biologic API,” states Michael Murray, director of downstream process development with Fujifilm Diosynth Biotechnologies. Each purification step in the downstream processing of biologic APIs serves a particular purpose, according to Turner. For monoclonal antibodies, he notes that Protein A chromatography effectively separates the product from most process-related impurities. Anion-exchange chromatography, meanwhile, is an excellent method for reducing DNA levels. For some products, hydrophobic-interaction chromatography can reduce aggregates to acceptable levels. Nanofiltration is typically included in modern purification processes as a dedicated viral clearance step.

	“If a process is challenged to obtain multiple logs of clearance of an impurity, then multiple removal steps are often required not only to meet the needed quality attributes but also to ensure process robustness, which leads to product consistency,” Murray observes. If the demand for clearance is lower, he notes that a single step can often be designed to ensure that quality attributes are met, but process robustness must be built into the step, either through thoughtful design or characterization of the design space.

	Advances in chromatography technology have been crucial to the improved performance of downstream purification operations. “Chromatography manufacturers continue to increase their offerings of different functional groups and bead structures from which a process development scientist can choose. For instance, resins are now available with increased capacity, improved selectivity, and tolerance to a wider range of conditions (i.e., salt tolerance),” Murray notes. A wider range of membrane chromatography options are also available that are enabling more optimized purification processes.

	There remains, however, growing pressure to increase the throughput of downstream processes to match upstream improvements in productivity and cell-culture titers. “The need is particularly true for monoclonal antibodies that are approaching commercial manufacturing. The challenge for development, engineering, and manufacturing will be to create unit operations that consistently achieve the desired quality attributes for a given step, but at a higher output,” says Murray.

	Further advances in downstream purification technologies could have positive operational and financial impacts and also influence biologic drug substance quality and consistency, according to Turner. These advances include low-cost alternatives to Protein A chromatography (e.g., synthetic affinity ligands), high-capacity membrane adsorbers to replace column resins, spray drying or bulk lyophilization of APIs, and room-temperature storage of APIs for increased shelf life. Murray adds that the combination of multiple unit operations into a continuous mode shows promise for improving throughput while still maintaining quality.

: 
	When referring to this article, please cite as C. Challener, Upstream and Downstream Operations Can Impact Biologic API Uniformity," 

Effective harvesting and purification processes play an essential role in ensuring that biopharmaceutical manufacturing processes provide biologic drug substances with uniform and consistent properties.

Ensuring Biologic API Uniformity

	Scientists from Eli Lilly and Company and Vanderbilt University reported the conversion of a batch-based stereoselective aza-Henry reaction between an arylnitromethane and Boc-protected aryl aldimine using a homogeneous BrÃ¸nsted acid-base catalyst to an automated intermittent-flow process with product crystallization and slow reagent addition (7). The organocatalytic enantioselective reaction included recycle of the catalyst, resulting in increased process intensity maintaining a high catalyst concentration in the reactor. The researchers performed a continuous campaign with the automated system and obtained high throughput and high selectivity while minimizing catalyst usage.

	Researchers at AztraZeneca, in combination with various European university groups, have focused on the development of continuous Buchwald-Hartwig reactions. In one report, the scientists used the highly active palladium N-heterocyclic carbene (NHC) precatalyst [Pd(IPr*)(cin)Cl] 4 (IPr* = 1,3-bis(2,6-bis(diphenylmethyl)-4-methylphenyl)imidazol-2-ylidene; cin = Î·3-cinnamyl), a universal approach for continuous workup and purification, and a methodology for catalyst recycling and reuse (9). Before implementing the flow reaction, the catalyst’s stability in water was investigated and an effective workup methodology was developed that allows separation of the Buchwald-Hartwig product into the aqueous stream as a salt while retaining the aryl bromide starting material in the organic stream with the catalyst.


	The conversion of low-temperature batch reactions to continuous processes is attractive because in many cases higher temperatures can be used to achieve equal or better results. For example, scientists from Eisai report the continuous production of eribulin mesylate via the diisobutylaluminum hydride reduction of an ester followed by n-butyllithium-mediated coupling of the resultant aldehyde to a sulfone fragment to afford a sulfone-diol as a key intermediate, all without the need for cryogenic conditions (11). The reduction was run at -50 Â°C instead of -70 Â°C, yet a better selectivity and yield were obtained, while the butyllithium-mediated coupling when run under flow conditions at 10 Â°C proceeded with a better conversion rate than the batch reaction when performed at -70 Â°C.

. 20 (2), 376-385 (2016).
	6. S. Borukhova, T. NoÃ«l, and V. Hessel, 


	When referring to this article, please cite as C. Challener, “Steps Closer to the Adoption of Continuous Processing," 

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