A rational approach to improving titer in Escherichia coli‐based cell‐free protein synthesis reactions

Cell‐free protein synthesis (CFPS) is an established method for rapid recombinant protein production. Advantages like short synthesis times and an open reaction environment make CFPS a desirable platform for new and difficult‐to‐express products. Most recently, interest has grown in using the technology to make larger amounts of material. This has been driven through a variety of reasons from making site specific antibody drug conjugates, to emergency response, to the safe manufacture of toxic biological products. We therefore need robust methods to determine the appropriate reaction conditions for product expression in CFPS. Here we propose a process development strategy for Escherichia coli lysate‐based CFPS reactions that can be completed in as little as 48 hr. We observed the most dramatic increases in titer were due to the E. coli strain for the cell extract. Therefore, we recommend identifying a high‐producing cell extract for the product of interest as a first step. Next, we manipulated the plasmid concentration, amount of extract, temperature, concentrated reaction mix pH levels, and length of reaction. The influence of these process parameters on titer was evaluated through multivariate data analysis. The process parameters with the highest impact on titer were subsequently included in a design of experiments to determine the conditions that increased titer the most in the design space. This proposed process development strategy resulted in superfolder green fluorescent protein titers of 0.686 g/L, a 38% improvement on the standard operating conditions, and hepatitis B core antigen titers of 0.386 g/L, a 190% improvement.

cells are lysed and the extract clarified. In both systems, this extract is then combined with a concentrated reaction mix containing nucleotides, amino acids, energy substrates, salts, molecular crowding agents, polymerases, and genetic material for the expression of the product of interest. Cell extracts have been generated from a variety of host organisms: archaea, E. coli, yeast like Saccharomyces cerevisiae and Pichia pastoris, mammalian cells like Chinese hamster ovary, HEK293, and HeLa, wheat germ, and tobacco. [4][5][6] In this study, we used E. coli-based CFPS; in addition to being one of the more economical options, it is the most wellstudied cell extract with dozens of publications to date and several commercial kits available on the market.
Transcription and translation are more streamlined in CFPS reactions because energy resources no longer need to be used to maintain metabolic activity for cell growth, though the protein synthesis rate is 200-fold slower than the in vivo rate. 7 The reactions are much shorter than in vivo cultivations, typically taking only a few hours. Titers of 2.3 mg/ml deGFP have been achieved with an E. coli-based CFPS system in batch mode in 10 hr. 8 Also, due to the absence of compartmentalized cells, CFPS reactions are open to additional components, like chaperones and detergents, which can easily be supplemented into the reaction to assist in protein folding and post-translational modifications. 9,10 When combined with orthogonal transfer RNAs, elongation factor Tu, and E. coli strains where release factor 1 has been eliminated, this open environment allows for the incorporation of nonnatural amino acids while maintaining high protein titers. 11 Several different formats have been used for the CFPS reaction including batch, fed-batch, microfluidic devices, and continuous-exchange reactions. [12][13][14][15] These reactions have been demonstrated to scale linearly in batch mode from the submilliliter scale to 100 L, a desirable characteristic for both the scale-up and scale-down of reactions. 12 Currently, Sutro Biopharma Inc. utilizes CFPS to manufacture an antibody drug conjugate with a nonnatural amino acid to allow for targeted conjugation to a specific site; the product is in clinical trials at present. 16 Recent work on lyophilized CFPS reactions has resulted in the development of "just-add-water" technologies, portable bioassays, environmental sensors, and paper-based sensors. 17,18 CFPS reactions have also been used for metabolic engineering and to observe gene circuits. 18,19 Though CFPS has many advantages and applications, little work has been done on process development for these reactions. Current process development strategies for a typical biopharmaceutical in a mammalian cell host involves lengthy cell line development schemes that can take 60-90 days. 20 While this process may be shorter for microbial hosts like E. coli and P. pastoris, it is still cumbersome and requires cloning and selection from several strains. The fact that CFPS reactions can produce good titers in a shorter timescale and do not necessarily require any cloning steps, as PCR products can be used, allows for the use of high-throughput methodologies to design a process development strategy. 21 We designed a process development strategy by examining the impact of process parameters, in particular cell extract strain, on product titer. Certain E. coli strains that have been engineered to have more stable mRNA or more eukaryotic tRNAs may result in better translation and increased yields for certain products. Similarly, we have observed the impact of induction on product titer in isopropyl β-D-1-thioglatopyranoside (IPTG)-inducible BL21 Star™ (DE3) E. coli cells. Induction of the E. coli cells prior to extract preparation should result in an increased concentration of T7 RNA polymerase, an enzyme vital to transcription. Adding T7 RNA polymerase into the CFPS reaction is commonly found in the literature, as T7 RNA polymerase is highly selective for its own promoter sequences and it has a transcription rate that is higher than endogenous E. coli RNA polymerase. 22 Because CFPS reaction conditions are no longer constrained by the needs of maintaining metabolically active cells, CFPS reaction parameters can be extended to greater extremes which may aid in the synthesis of difficult-to-express proteins and self-assembly processes (e.g., virus-like particles [VLPs]). [23][24][25] By adding more of the potentially limiting components for example, plasmid DNA or cell extract, the reaction equilibrium may be shifted to increase product yield. However, as both plasmid and extract preparation are time-consuming and labor-intensive processes, it is critical that the system not use more than what is required for either component. As the reactions are no longer limited by cell growth, the pH of the concentrated reaction mix can be decreased or increased far beyond typical physiological levels.
However, pushing the pH of the reaction too far may have an adverse effect on the molecules required for transcription and translation of the product (ribosomes, polymerases, enzymes, and so forth) or could result in the precipitation of other reaction components. Because the polypeptide elongation rate in E.coli cells is enhanced at higher temperatures, increasing the reaction temperature should increase production, though too much of an increase may lead to protein degradation. 26 Additionally, lower temperatures may be beneficial as CFPS reactions are considered less thermostable than their corresponding host cells because they are more dilute; lower temperatures may also aid in protein folding and prevent aggregation. 27 The length of the reaction should be long enough to allow for the expression of high titers of protein but should not be so long that inhibitors like inorganic phosphate saturate the system. 28 The work presented here investigates the impact of the aforementioned process parameters on product titer. We examined the E. coli strain used for the cell extract, two compositions of the concentrated reaction mix, and plasmid selection. We used multivariate data analysis (MVDA) to generate a model based on the titers resulting from reactions where the plasmid and cell extract concentration, pH of the concentrated reaction mix, reaction temperature, and length of reaction were manipulated individually. (We chose to compare two concentrated reaction mixture rather than investigate the individual components of the concentrated reaction mixtures because this has already been examined in some depth elsewhere. 29,30 ) By performing a multilinear regression (MLR), we predicted which combination of parameters result in the highest titers within the robust operating space defined. The process parameters with the largest influence on titer were further evaluated through a response surface design of experiments (DoE) approach enabling the operating conditions that led to a significant increase in product titer to be identified. Several other groups have used DoE previously to examine multiple parameters at once while using a minimal amount of reaction material in order optimize parts of the CFPS system including extract preparation, chaperone and salt concentrations for expression of proteins with disulfide bonds, and the ratio of heavy chain expressing plasmid to light chain expressing plasmid for antibody expression. 12,[31][32][33] Here we use DoE for titer maximization in a given design space, rather that optimization, to demonstrate how this process development strategy might be used with two different proteins.

| MATERIALS AND METHODS
All chemical reagents were purchased from Sigma-Aldrich (Dorset, UK) unless otherwise stated.

| Extract preparation
The extracts were derived from the BL21 (DE3) (Thermo Fisher Scientific, Paisley, UK), BL21 Star™ (DE3) (Thermo Fisher Scientific), and Rosetta™ (DE3) E. coli strains using the method outlined previously. 34 Briefly, a small volume, approximately 100 μl, of bacterial glycerol stock was used to inoculate 50 ml fresh Lysogeny broth (LB) medium (pH 7.4) in a 250 ml baffled shake flask. The cultures were incubated overnight at 34 C and 250 rpm. The following day, approximately 16 hr later, 25 ml of the overnight culture was transferred to 500 ml of 2×YTPG medium pH 7.2 (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, 7 g/L K 2 HPO 4 , 4.3 g/L KH 2 PO 4 , and 18 g/L glucose; adjusted pH to 7.2 with potassium hydroxide) in a 2 L baffled shake flask. The culture was incubated at 34 C and 220 rpm until OD 600 ≈ 2 was achieved at which point 500 μl of 1 M potassium hydroxide was added to prevent acidification of the culture (as recommended by Hong et al. 2015) and the incubation continued. 34 When OD 600 ≈ 4 was achieved, the culture was harvested by centrifugation at 5,000 g and 4 C for 15 min; the cells should be entering stationary phase.
Contrary to popular methods of harvesting during the mid-late log phase, Failmezger et al. showed that high performing extract can be produced from E. coli in the stationary phase; as such, we decided to simplify our workflow and adopt their method of extract production. 35 The supernatant was discarded and the pellets were kept on ice whenever possible. Each pellet was washed with~25 ml of S30 buffer (pH 8.2 10 mM Tris acetate, 14 mM magnesium acetate, 60 mM potassium acetate, and 1 mM dithiothreitol) and resuspended by vortex. The resuspended cells were pelleted by centrifugation at 9,000 g and 4 C for 10 min. The pellet was washed, resuspended, and pelleted by centrifugation again. Excess supernatant was discarded.
Pellets were stored at −80 C following this step. Pellets were resuspended in 1.0 ml of S30 buffer per 1.0 g of pellet. The pellet was thawed on ice with S30 buffer for at least 1 hr prior to resuspension.
The resuspended cells were homogenized via single pass at 1,000 bar through an APV Gaulin Micron Lab40 Homogenizer (Lubeck, Germany). The homogenized lysate was clarified by centrifugation at 30,000 g and 4 C for 30 min. The supernatant was recovered and centrifuged again using the same conditions. The supernatant from the second centrifugation step was decanted and separated into 1 ml and 200 μl aliquots. Aliquots were flash frozen with liquid nitrogen and stored at −80 C until use.
The extract preparation method was not optimized or varied for different strains, as the authors have seen no evidence of any strain or product specific impact from extract preparation methods discussed in the literature. While we chose this protocol based on the equipment and reagents at our disposal, we recognize that numerous groups have thoroughly examined the extract preparation protocol and optimized conditions, such as length of time for cell growth, length of time for induction, and media for cell culture. 31,[36][37][38][39] In addition to the extracts prepared above, another BL21 Star™ (DE3) extract was prepared following the protocol above with the addition of 500 μl of 1 M IPTG when the 500 ml shake flask cultivation had achieved OD 600 ≈ 0.6. This induction was done to increase the amount of T7 RNA polymerase in the extract. Unlike the other strains, these cells were incubated at 37 C.
A Bradford assay was used to determine total protein concentration for each extract; all extracts gave values of 30-50 mg/ml as expected based on the previous literature. 40,41 The total protein concentrations were as follows: 38 mg/ml for BL21 Star™ (DE3), 35 mg/ml for the BL21 (DE3), 34 mg/ml for Rosetta™ (DE3), and 44 mg/ml for the IPTG-induced BL21 Star™ (DE3).

| CFPS reaction
A cell-free concentrated reaction mix based on the protocol used by Kwon and Jewett (2015) was prepared. 40 The cell-free reaction

| Adjusting continuous process parameters
Each of the following process parameters was examined in isolation in order to determine their impact on titer: plasmid concentration, amount of extract, temperature, pH of the concentrated reaction mix, and length.

| Design of Experiments
Following the "one-variable-at-a-time" analysis detailed in the previous section, a response surface DoE study was performed for each product to better understand the interactions between different process parameters and the subsequent impact on titer. This exercise was done to demonstrate how titer might be maximized using response surface DoE and validate its use as part of this process development strategy. It is not the intention to optimize titer, though that could be done by expan-

| GFP plasmids
Commercial CFPS kit suppliers recommend using a plasmid which has been optimized for cell-free expression though plasmids typically employed for cell-based expression can be used as well. 44 Two GFP plasmids were selected: pJL1, a superfolder GFP plasmid, pJL1 was a gift from Michael Jewett (Addgene plasmid # 69496; http://n2t.net/ addgene:69496; RRID:Addgene_69,496), and pET14b-GFP, a plasmid developed by Martin Warren's group at the University of Kent used for E. coli expression of GFP+ with a 6xhistidine tag. Plasmid pJL1 has been optimized for CFPS. It is a much smaller plasmid (2,486 bp) that contains only the gene of interest (sfGFP in this case), the T7 promoter, the T7 terminator, a gene for kanamycin resistance and an origin of replication. The pET14b-GFP plasmid has not been optimized for cell free and has not been codon-optimized for E. coli. It is also somewhat large compared to the pJL1 plasmid, 5,389 bp.

| GFP analysis
For GFP analysis, it is assumed that all GFP proteins that have been produced are correctly folded and emit with the characteristic fluorescence intensity for that GFP variant. Titer was measured through fluorescence intensity measurement on a BMG Labtech (Aylesbury, UK) FLUOStar OPTIMA spectrophotometer at an excitation wavelength of 485 nm and an emission wavelength of 520 nm and compared to a standard curve of rTurbo GFP from Evrogen (Moscow, Russia). The range of the standard curve was 80-1.6 μg/ml. To dilute into this range, all CFPS reactions were diluted 10-fold (20% vol/vol non-induced BL21 Star™ cell extract diluted in reverse osmosis water) For different GFP variants, fluorescence intensity was scaled based on quantum yield and extinction coefficient using the following equation where "F" is the measured fluorescence of a sample, "φ" is the quantum yield for that variant, "I 0 " is the intensity of the incident light, "ε" is the extinction coefficient for that variant, "l" is the optical path length, and "c" is the concentration of a sample.

| HBcAg plasmid
The plasmid for the HBcAg dimer was obtained from iQur Ltd. 49 It is a plasmid for monomeric HBcAg subtype ayw under the T7 promoter in a pETDuet-1 backbone. Its exact sequence is unknown, but it is 5,900 bp long. This plasmid has been used previously in vivo; it was not optimized for CFPS.

| Multivariate data analysis
MVDA was used to evaluate the results from the reactions detailed in Materials and Methods (Section 2) to determine which combination of conditions would maximize titer and to gain a better understanding of each variable's contribution on titer for both products investigated.
Data manipulation and analysis for the MLR model was performed using MatLab R2019b (MathWorks, Inc., Natick, MA). MLR was used to predict a single dependent variable-titer-from a series of independent inputs-plasmid concentration, amount of E. coli extract in the reaction, pH of concentrated reaction mix, temperature of reaction, and length of reaction. In this manner, the contribution of each independent parameter on titer can also be determined. The variables of importance were found by creating multiple MLR models that studied the influence of each parameter that was removed during the development of models that considered linear, quadratic, polynomial (squared terms and cubed terms), and interactions. Separate models were created for sfGFP and HBcAg titers. The prediction performance of the MLR was quantified using the coefficient of determination, which is calculated as: where, y i is the product concentration for run i, y is the product concentration mean, andŷ i is the predicted product concentration for run 3 | RESULTS AND DISCUSSION

| Establishing a working CFPS system
In order to increase titer in our CFPS system, we first need to design a CFPS system to establish a baseline. We considered three major com- were analyzed based on sfGFP and GFP+ production.
The reactions with the pJL1 plasmid achieved over double the titer achieved with the reactions using the pET14b-GFP plasmid: an average of 227 μg/ml compared to an average of 106 μg/ml ( Figure 1a). This demonstrates that traditional plasmids may not perform as well as a plasmid optimized for CFPS. Care should be taken, however, not to assume that this is generally true. [50][51][52][53] The pJL1 plasmid expresses sfGFP, which folds more readily and is brighter than the GFP+ produced using the pET14b-GFP plasmid. 54,55 Though we have corrected for this, the fluorescence readings for sfGFP are generally stronger. In addition, using plasmid preparation kits, we are usually able to produce more pJL1 plasmid (~400-500 ng/μl,~240-300 nM) than pET14b-GFP plasmid (~150-250 ng/μl,~40-55 nM).
We  40 and the pJL1 plasmid. Error bars represent plus or minus one SD for n = 3 biological replicates, each represented as a single data point. In typical reactions, the concentrated reaction mix was combined with 6.1 nM (10 μg/ml) pJL1 plasmid and 20% vol/vol cell extract and then incubated at 30 C for 4.0 hr. CFPS, cell-free protein synthesis sources in the minimal mix; the complex mix contains more energy sources, in particular PEP, CoA, and NAD + , that may allow for prolonged ATP regeneration. 30 The complex mix also utilizes nucleotide triphosphates instead of nucleotide monophosphates, as used in the minimal mix, which may allow for better ATP regeneration as well as higher rates of transcription and translation, especially when paired with additional E. coli tRNAs, which are also absent from the concentrated minimal mix. However, it is worth noting that CFPS reactions have been shown to be able to generate nucleotide triphosphates from nucleotide monophosphates in both crude cell lysate CFPS and the PURE system. 29,57 Based on these results, the complex concentrated reaction mix was used in subsequent screening studies.
Then, we examined three different E. coli strains: BL21 (DE3), BL21 Star™ (DE3), and Rosetta™ (DE3) ( Table 1). 58 The BL21  The BL21 (DE3) Star™ strain, as with all the DE3 strains used in this study, can be induced with IPTG to produce T7 RNA polymerase. 59 We therefore investigated the impact of IPTG induction during cell cultivation on cell extract performance. While constitutive promoters like σ 70 promoter can be used in CFPS, the gene of interest is under the T7 promoter in all the DNA plasmids used in this study. 35,60 Thus, T7 RNA polymerase results in the expression of the target gene.

| Comparison to a commercial CFPS system
Overall, the combination of extract, concentrated reaction mix, and plasmid that gave the highest average titer of sfGFP was the BL21 Star™ (DE3) extract, the complex concentrated reaction mix based on the protocol in Kwon and Jewett (2015) and 6.1 nM (10 μg/ml) of the pJL1 plasmid. This CFPS platform was compared to the commercial kit sold by ThermoFisher Scientific™, the Expressway™ Mini-Cell Free Expression System. 44 The reactions were analyzed based on sfGFP production. We found this platform, which gave an average titer of 497 μg/ml, performed as well as the commercial kit, which gave an average titer of 493 μg/ml (data not shown).

| Confirming the choice of extract for the second product: HBcAg
Now that we have established our CFPS system, we can manipulate various process parameters to improve product concentration and use this analysis to inform our process development strategy. We have chosen to demonstrate this strategy with two products, sfGFP and HBcAg VLPs. We have already determined the appropriate E. coli strain for the cell extract for sfGFP production, and before we can employ our process development strategy, we will need to do the same for HBcAg.
HBcAg production was observed in CFPS reactions with the four previously mentioned cell extracts (Figure 3a). We found that an Amount of extract from 5% vol/vol to 35% vol/vol was examined with sfGFP and 5% vol/vol to 30% vol/vol with HBcAg. Titers were relatively consistent (~450 μg/ml sfGFP and~175 μg/ml HBcAg) when amount of extract was above 20% vol/vol (Figure 4b). It is likely that other resources (polymerases, amino acids, nucleotides, and so forth) are depleted and plasmid or extract is no longer the limiting reagent or that inhibitors like inorganic phosphate have accumulated.
Commercial kit suppliers recommend reaction temperatures between 30 and 37 C. 44 We expanded this range testing reactions at temperatures from 15 to 40 C for sfGFP and 20 to 35 C for HBcAg.
Titers peaked with a reaction temperature of 32-35 C for both products ( Figure 4c). Though 32-35 C may maximize sfGFP and HBcAg production, other products may require higher or lower temperatures. Lower temperatures might be preferable for more complex molecules with solubility issues as this tends to reduce the formation of inclusion bodies. 64 Previous studies have indicated that pH is one of the most critical process parameters in CFPS reactions. 41,65 In sfGFP production, we also observed this to be true; as the pH of the concentrated reaction mix decreased, the product concentration increased (Figure 4d). Titers of over 700 μg/ml were achieved with a concentrated reaction mix of pH 5.5. This is likely because the other components in the CFPS Reaction length is highly variable amongst previous studies: batch reactions from 2 to 24 hr have been examined. 28,68 In our own studies, we observed a visible green tint to the CFPS reactions producing sfGFP after only 0.5 hr of incubation; therefore, we examined reaction lengths from 0.5 to 22 hr for sfGFP and 0.5 to 24 hr for HBcAg. In observing the length of the reactions, titers stabilized after 4 hr for both products (Figure 4e). However, as the length of the reaction increases, the variability in titer becomes much greater. Therefore, when possible shorter reaction times are recommended. Also, sfGFP is known to fold efficiently with good folding kinetics. 54 Other products with known assembly issues may require longer reaction times. Alternatively, certain amino acids and nucleotides may be depleted after 4 hr of reaction. To replenish these reagents, a concentrated solution of amino acids and nucleotides could be fed into the reaction or continuous reactions could be used instead of the batch method employed here. 69 3.5 | MVDA to maximize product titer It was difficult to quantify the influence of each variable on product concentration due to the complex interactions between all process parameters. Therefore, to quantify the relative importance of each MVDA was selected to evaluate the screening design based on its proven ability within the biopharmaceutical sector to leverage useful information from complex data sets and uncover useful correlations that are not always obvious from univariate analysis. 70 The DoE methodology implemented is a systematic approach enabling the relationship between process operation and process output to be determined while reducing the required number of experiments to understand these key relationships. The face-centered composite DoE was selected as it is the most appropriate design when factors investigated cannot be extended beyond the factorial points which was the case in this experiment. It also enables linear, interactive, and quadratic terms to be evaluated as it contains center points in addition F I G U R E 5 Multivariate data analysis of process parameters. The MLR models for sfGFP and HBcAg titers, respectively, are shown in (a) and (b), where each bar represents the product concentration from an experimental run (the experimental runs are the same ones shown in Figure 4 and detailed in Table 2 and Table 3). The contributions of each parameter to titer is shown in (c) and (d), where "Time" is the length of the reaction "Plas" is the plasmid concentration, "Ext" is the amount of extract in the reaction, "pH" is the pH of the concentrated reaction mix, and "Temp" is the temperature of the reaction. T A B L E 2 Experimental conditions for reactions producing sfGFP used for MVDA in Figure 5(a) T A B L E 3 Experimental conditions for reactions producing HBcAg used for MVDA in Figure 5(b) Step 1 (extracts explored): BL21 (non-induced) Rosetta (non-induced) BL21-Star (non-induced) BL21-Star (IPTG-induced) Step 2 (ranges explored):

BL21-Star (IPTG-induced)
Step The maximum titer for both products is located at the edge of the design space suggesting titer could be further improved and optimized by widening the experimental design space considered in this work.
However, it is not our intention to optimize titer in this work, merely to demonstrate the advantages of our systematic process development approach, as summarized in the following section, and to maximize titer within a given design space. It is also important to mention that expanding the design space could have other unintended consequences. For example, the optimum titer of HBcAg expression may be achieved with increased amounts of plasmid and extract, but significantly increasing the presence of these components in the reaction also increases the cost of the reaction.

| CONCLUSION
CFPS allows for recombinant protein production over short reaction times in small reaction volumes; we are therefore able to deploy a process development strategy that could be completed in under 48 hr. We determined that the most critical parameter is the E. coli strain chosen for the cell extract. Beyond that, titer can be incrementally increased by manipulating other process parameters. This method can be easily applied to new or difficult-to-express products in addition to efficiently testing a wide range of reaction conditions.
We recommend first examining a series of extracts from different E. coli strains and observing the effects of IPTG-induction where possible before conducting further experiments on other parameters ( Figure 6). Subsequently, MVDA modelling should be applied to determine the influence of process parameters on production. Then, a DoE study should be used to identify the process conditions that result in the highest titer in the design space. Using this strategy, we were able to increase sfGFP and HBcAg titers by 38 and 190%, respectively, beyond the standard conditions. We recommend this method over an initial scouting DoE due to the multitude of parameters that can be manipulated in CFPS reactions. Here we examined extract strain, concentrated reaction mix composition, plasmid selection, plasmid concentration, amount of extract, pH of the concentrated reaction mix, reaction temperature, and length of reaction. Depending on the product, other parameters like chaperone concentration, agitation rate, T7 RNA polymerase concentration, osmolality of the concentrated reaction mix, or protease inhibitor addition could also be critical for improving titer. While an initial DoE study to examine all these parameters could easily be performed given the high-throughput nature of CFPS, many parameters would not be critical to improving titer and the CFPS reactions in which those parameters are manipulated would be a waste of time and resources. By using MVDA to determine the parameters with the highest influence on titer, we can rule out other parameters and take a DoE approach that focuses only on the most critical parameters.

ACKNOWLEDGMENTS
We would like to acknowledge Mark Turmaine for his assistance with

CONFLICT OF INTEREST
W. R. is the CEO of iQur Limited.

PEER REVIEW
The peer review history for this article is available at https://publons.