Genotoxic Impurity Considerations

Genotoxicity is the capacity of a substance to damage genetic material such as DNA, and thus cause mutations or possibly cancer. Genotoxic impurities need to be avoided in a pharmaceutical product because they can possibly cause serious health consequences even at levels less than standard analytical methods can detect.

It has been argued that most genotoxic reagents/intermediates present in syntheses that have four or more stages prior to API isolation/purification are likely to be deactivated by reaction with other reagents or purged by dissolution in solvents, removed by vacuum distillation or other phase switching operations; however, regulatory reviewers who have a vested interest in emphasizing the seriousness of genotoxins show little sympathy for such arguments. Government reviewers tend predominantly to request quantitative analytical information on the API and/or intermediates possibly combined with the results of spiking experiments (impurity fate analysis) and could well require these in order to demonstrate the absence of carryover of potential genotoxic impurities (PGIs), at a suitably low (TTC) level. Such investigations can be highly resource-intensive and challenging, particularly in respect of developing validated analytical methods for low levels of multiple PGIs. Moreover, limits for certain PGIs may become part of the API specification and so become an ongoing quality-control commitment.

However, concerns usually relate to late-stage operations that involve potential genotoxic impurities PGIs. One EU review raised as a major objection, potential residues of mesityl oxide (4-methyl-3-penten-2-one) in a drug substance crystallized from acetone.  Another cited possible traces of alkyl mesylates (methyl, ethyl and isopropyl mesylates) in a mesylate salt drug substance.

Unless the threatened concern about the PGI is identified at the early planning stage, modifying a route that is otherwise the best proposal should be the last resort.  Nonetheless, modification of the API synthesis in a way that minimizes PGI levels in the drug substance has been undertaken in some cases; recent published examples pertain to formaldehyde, chloroalkanes and acetamide.

Given the difficulties in obtaining reliable predictions, particularly for aromatic amines, it may be prudent to undertake an Ames assay on any PGI containing an aromatic amine structural alert, if data are not already available in the public domain. Aromatic amines that otherwise make admirable process intermediates because of the multiple options for their purification, should where it is feasible be transformed to more acceptable functionalities early in a process route.

The potential action of small alkyl alkylating agents presents a particularly frequent and acute problem for pharmaceutical products because so many of them in their API salt form are hydrochlorides, methanesulfonates, tosylates and besylates as well as to a much smaller extent, hydrobromides.

These pharmaceutical salts are most often prepared in the final chemical transformation of a sequence and so any PGI has the highest probability of lingering behind in the API.

The best evidence that these concerns about PGIs arising from making the aforementioned pharmaceutical salts are without any foundation is the epidemiological evidence of the health of medicinal and process chemists who have been making and using these salts for years and years without any relative health damage.

The apparent lack of health damage is readily explained since the reactivity of halo compounds in biological systems can be predicted to a significant extent on the basis of their relevant chemical properties such as alkylating potential and susceptibility to hydrolysis. Thus, bromo compounds are expected to be more reactive than chloro compounds. SN1 and SN2 characteristics will determine the nature and the extent of reactivity towards nucleophiles, and steric factors may also play a part in some cases. In the 4-(p-nitrobenzyl)pyridine alkylation assay, alkyl halides generally show negligible activity, methyl methanesulfonate (MMS) being at least 40 times more active than ethyl, propyl or butyl bromide. Allyl bromide appears to be more active (around one-eighth of the activity of MMS although allyl chloride shows minimal activity. Benzyl chloride, while rather active for a chloro compound, is around 20-fold less active than allyl bromide. Owing to their volatility and/or hydrophobicity many alkyl halides show negative results in conventional Ames Salmonella assays, and it is often necessary to employ vapour phase exposure in a closed system (using a desiccator for example) in order to obtain positive results. Most alkyl halides, especially bromides, are Ames positive (using a closed test system if necessary), although 1-chloropropane, 1-chlorobutane and neopentyl bromide are all Ames-negative. As expected, based on their lack of alkylating activity, both chloro- and bromobenzene are Ames-negative. Some unsaturated halo compounds have the potential to be metabolized to form quite active mutagenic molecular species. For example, evidence suggests that oxidative biotransformation of vinyl chloride produces chloroethylene oxide and 2-chloroacetaldehyde as active metabolites. Binding of bromobenzene-3,4-oxide to liver proteins is thought to account for the hepatotoxicity of bromobenzene. The predominant metabolic pathway for simple alkyl halides is halide displacement by glutathione, although some C-hydroxylation reactions may occur.

Rodent bioassay data on alkyl halides strongly suggest that these compounds are either non-carcinogens (1- chlorobutane, bromomethane) or low-potency carcinogens (chloroethane, bromoethane). Both chloroethane and bromoethane produced an increased incidence of a rare type of endometrial tumour in female mice and it seems highly plausible that the carcinogenic effect is caused by a species-/ gender-specific stress-related adrenal overstimulation and excessive corticosteroid production. The rodent carcinogenicity profile for chloroethane (increased incidence of a rare tumour type at an extremely high concentration of 15,000 ppm in one species/gender) is thus much closer to that for a non-genotoxic carcinogen than for a genotoxic carcinogen. Thus, the (feeble) alkylating activity of chloroethane seems largely incidental to its carcinogenic activity, a scenario likely to apply to many other similar alkyl halides. This prediction is strongly supported by the fact that benzyl, ethyl, isopropyl, and trityl bromides were inactive as carcinogens at doses up to 0.83, 12.5, 8.3, and 0.25 mmol/kg, respectively, when administered by single subcutaneous injection to female rats. A number of independent expert assessments are available on halo compounds.

Acceptable/tolerable exposures are expressed in various ways, for example as minimal risk levels (MRLs) by the Agency for Toxic Substances and Disease Registry, or as reference concentrations/doses (RfCs/RfDs) by the U.S. Environmental Protection Agency. There is a clear consensus that chloroethane is less hazardous than the more reactive chloromethane, although recommended safe exposures for the former range from the highly conservative OEHHA value of 150 μg/day to 200 mg/day (10 mg/m³ at an average air intake of 20 m³/day⁴³) based on the EPA IRIS assessment.

 Acceptable exposures in the context of genotoxic impurities can also be calculated on the basis of the TD50 values as described above, resulting in PDEs of 1810 and 149 μg/day for chloroethane and bromoethane respectively. A PDE for non-carcinogenic 1-chlorobutane could be determined using ICH Q3C (R3) methodology on the basis of the most appropriate NOAEL in lifetime studies.

Quotes from Org. Process Res. Dev. 2011, 15, 1243–1246

[Compounds can be grouped together that have] strikingly different reactivities, isopropyl chloride and isopropyl mesylate for example. Isopropyl chloride is Ames-negative in assays using standard conditions; testing has to be carried out in a desiccator to obtain a (feebly) positive result; on the other hand, isopropyl mesylate (Swain-Scott s = 0.29) is a potent mutagen in the standard Ames test and gives positive results in several in vivo assays. Hydrolysis half-lives at pH 7.0 and 25 C are 38 days (the same as for chloroethane; TD50 1810 mg/kg/day) and 4.5 h, respectively. Unfortunately, no rodent bioassay data appear to be available for either compound, although 1,2- dichloropropane is Ames-positive in standard assays and has a mouse TD50 of 276 mg/kg/day (negative in the rat). A Risk Specific Dose (RSD) for isopropyl chloride of approximately 37 μg/day (for a 50 kg patient) has been determined by Bercu et al. and Contrera based on QSAR (quantitative structure- activity relationship) techniques using regression analysis of “training sets” of TD50 data. [The choice of compounds in the isopropyl chloride training set could be questioned in that it contained several non-genotoxic polychloro compounds but not 1,2-dichloropropane or chloroethane.] Use of QSAR models to predict genotoxic/carcinogenic potency relies on rule-based techniques (such as DEREK) or statistical techniques (using regression analysis of training data sets), and particularly in relation to carcinogenicity, the latter approach is considered to provide more reliable results. Since halo compounds in particular and other compounds containing “Ashby alerts” are often identified as PGIs, it is interesting to note that in the determination of the TTC limit Kroes et al. classified only two such compounds (5% of the total in the data set) in the lowest potency category (equivalent to 1.5 μg/day), but since the data set is non-transparent it is not possible to identify these two key compounds. [In a prior publication using essentially the same data set, the TD50 for methyl methanesulfonate is listed as 0.178 mg/kg/day, 179 times lower than the true value, and it is unclear whether this miss-transcription was carried forward to the data set used by Kroes et al. in the more “definitive” publication.] In the Kroes publication, no carbamates were classified in the lowest potency category, suggesting that the standard TTC of 1.5 μg/day may be unnecessarily conservative for this structural class.  

Diethyl Phosphite: A Solvent you wouldn’t easily think of -Miscible both with Water and Ordinary Organic Solvents

 

Diethyl Phosphite is variously called diethyl phosphonate, diethyl hydrogen phosphite, diethyl phosphonite. 

According to Fieser & Fieser Organic Reagents Vol. 1 pg. 252 “Diethyl phosphonate {diethyl phosphite/diethyl phosphonite/(EtO)₂P(=O)H} is miscible with water and with ordinary organic solvents and has remarkable solvent power.” Diethyl phosphite may be just the trick when all the ingredients for a reaction cannot be dissolved together!

 It is also recommended for the preparation of phenyl hydrazones and 2,4-dinitrophenylhydrazones in the short communication: Maynard, J.A. Australian Journal of Chemistry, 1962, vol. 15, p. 867 - 868. These latter reactions drive the equilibrium by removing water by reaction while generating a useful strong acid as catalyst. It is also important that diethyl phosphite readily dissolves both the reagent and substrate. Dr. Maynard points out that the addition of an acid catalyst to the mixture is unnecessary because the solvent reacts with any water initially present and all that is formed in the reaction to produce the strong acid ethyl hydrogen phosphate. Thus the needed acid is produced and the by-product water is scavenged. Many of the phenyl hydrazones made using this solvent precipitated directly; others required mixing with water equal in volume with the solvent. Recrystallizations were from methanol, ethanol, benzene, or acetone as the solubilities directed.

This trick of binding up by-product water and generating useful acid may apply to other reactions.

Increasing Throughput in Chemical Process Development by Reducing Cycle Time

 

 Reducing Cycle Time

Often the easiest way to improve throughput is to reduce cycle time and this can be done just as often working on the work-up, isolation, or purification sequences as changing the actual chemical reactions. 

By cycle time, I mean the time it takes to run a batch when the reactor, reactor accessories, and peripheral equipment, such as centrifuges, filters, driers etc. are deployed as continuously as possible making the end product of that step. This includes cleaning, verifying, noting the results of the previous run …everything that must be done. The goal of reducing cycle time is to compress more process step runs into the same amount of time.

Some chemical process steps contain operations in which solution or slurried reactor contents are concentrated by removing some volatiles. If this concentrating consumes a significant amount of time, making it more efficient might go a long way or even all the way towards increasing throughput by reducing the cycle time. 

In the fine chemical industry most evaporations are either semi- or fully batch. While the product from the evaporation step can either be the overhead or the pot contents, the liquid to be separated is usually either wide-boiling or has a non-ideal vapour/liquid equilibrium (such as an azeotrope), which results in a more discreet separation between mixture components. 

Processes where liquid concentration is a predominant aspect actually use special equipment to remove unwanted fluid. A typical industrial evaporator has tubular heating surfaces, a vessel to hold the charge and sweep away vapour from liquid, and a condenser (heat exchanger) to condense the lighter overhead fraction. These units can operate at atmospheric or elevated pressures, but are often run under vacuum to reduce the system temperature. This unit operation can be run continuously, semi-batch-wise, or fully batch-wise. 

Most multi-purpose plants do not have access to such equipment; however, even in a process that is predominantly chemical reactions, getting access to such specialized but common equipment may be advantageous if part of a workup or isolation is a laborious concentration.

In batch chemical reactors commonly encountered in the typical multipurpose fine chemical plant heating is commonly simply coming from the reactor walls. 

Still even here the operation of concentrating solutions or slurries by batch distillation, can often be shortened. In most cases, these practical approaches are applicable to both vacuum and atmospheric evaporators. With respect to the evaporation the question needs to be asked, “Where are we with respect to this system’s capacity?” 

If an important element of the problem is the rate of heat transfer from a jacketed reactor’s walls, injecting live steam into the reactor is another way to supply heat. By using what is rudimentary steam distillation the vapour fraction of the organic volatile which you are trying to remove is reduced-yes- but the actual rate of heat transfer and hence mass vaporized per unit time may more than compensate for that. So long as your condenser can handle it, steam distillation can be combined with reduced pressure to give vacuum steam distillation. Contrary to what you might think, distilling with steam may actually cause less degradation than ordinary distillation because the temperature of the steam may be much less than the temperature of the jacket’s walls.

Even if water has deleterious effects on the charge you are trying to concentrate, inert gas sparging can still speed up an operation. We know this because there are anecdotal reports that reactions that use volatile catalysts can stall on scale-up because the higher rate of inert gas sparging provided in the pilot plant unexpectedly reduced the charge of catalyst.

If the process is currently set up as atmospheric evaporation, the obvious change would modify the process train such that it can become a vacuum operation. The first and hopefully most obvious issue here is to ensure that all equipment in the evaporator train is rated for vacuum service. If not, this option may require a substantial capital investment and other means of achieving desired capacity may need to be investigated first. However, if the process is already configured for vacuum evaporation, it can simply be run at lower pressures, thus allowing the system to operate at lower temperatures, keeping the product cooler. 

Take into consideration that running at lower pressure may mean a decrease in vapour density and, thus, an increase in vapour loading to the condenser to achieve the increase! Ensure that the condenser and utility streams are capable of handling such an increase. 

Temperature Sensitivity Issues 

When the product is a solute, one needs to know the yield/product losses due to the heat-treatment of temperature-sensitive materials. Perhaps the recovery-yield can be improved by more precise temperature control and throughput increased just by raising the overall yield. Even if the system’s overall temperature is not deleterious to the product, ensure that local hot spots in the base heater are not degrading some material. One way to resolve this is to ask: "Can we run the process at a lower temperature (which usually means running at lower pressure), such that we can keep the product cooler?" Again, this may extend the cycle time, but if yield improvements are large enough to counteract those losses, this could be an elegantly simple change which alters throughput in the right direction. 

More Precise Heating with a Tempered Loop

An option that provides benefits both in temperature control and heat-transfer efficiency is to install what is called a tempered loop feeding your base heater on the liquid-service side of the heat exchanger. In a tempered loop, a pump re-circulates the bulk of the heat-transfer fluid through the reactor jacket at an increased rate, with a small bleed-in of cold or hot utilities to achieve accurate temperature control. The increased mass of re-circulating fluid buffers the temperature, improving temperature control, which, in turn, permits running a little closer to any limiting temperature values (examples might be the freezing point of the condensate mixture or degradation of a heat-sensitive material due to a minor control upset on the base-heater service fluid). Higher fluid velocities and a higher corresponding Reynolds number positively affect the fouling resistance, as well as the overall heat-transfer coefficients and, thus, the heat exchanger efficiency. 

However, with the tempered loop, as with all changes, there are checks that need to be made to ensure that the system works properly. Be careful that the heat exchanger is sized to handle increased flow rates, velocities, pressures and pressure drops. This is something where you need the input of a chemical engineer. It can be problematic, and maybe dangerous, that after changing to a tempered loop, the heat exchanger's pressure-relief setting and system pressure are too close, causing the system to relieve the pressure with any pressure spike. 

Accurate temperature control is an area to look at early on in evaluating capacity increases or when troubleshooting temperature problems (such as freezing condensate) in heat-transfer systems. For a relatively small investment, the reward can be large in terms of condenser freeze protection, additional heat duties and overheat protection needed for temperature-sensitive materials. 

Cleaning

In cleaning, some of the desired material is removed from the equipment and ends up in the waste. In flushing (treated further below) residual desired material is removed from the equipment and combined with the major recovered amount. 

Another area often overlooked when searching for more capacity, perhaps more related to specific process materials than the unit operation itself, is the cleaning cycle time. Assess the impact the cleaning cycle has on the overall cycle time and then investigate what changes can be made to simply reduce this time. Assess the appropriateness of the cleaning agents, analyze the cleaning sequence, the quantities used, etc. Optimize the water flush/rinse times or volumes to achieve more production time. Reducing the amount of idle time increases the number of lots processed.

When repeating certain steps with identical runs one after the other only a partial cleaning may be needed and only a visual inspection rather than the more thorough swabbing and chemical analysis. This saves a lot of time that was being taken into account measuring throughput.

Product Flushing

Particularly for batch or semi-batch processes, eliminating or minimizing physical product losses in between batches from inadequate flushing of precipitated materials can increase the recovery yield. Changing flushing techniques or solvents can do this. 

Whereas extending flush time may cut some capacity by extending cycle time, it may still be justified if there is sufficient increased recovery. 

Look closely at the product/solute properties. If the product is sticky, look for a flushing agent that is compatible with downstream processes but will aid in removing such particles prior to their transfer to the next process step. 

Another example where extra flushing may pay dividends is recovering residual products from charcoaling and other solid adsorbent treatments.

Top Ten Kilomentor Chemical Process Development Blog Articles

The Kilomentor Blog has existed for seven years. It was originally hosted on Chemical Blogs but for the last three years, it has been a Google Blogspot tenant. Although, as the editor, I can see which of my blog articles has received the most readership, this is not available to readers.

Readers do have access to an in the blog search engine that can select articles pertinent to the keywords they select; however, I thought it might interest all readers to have links to the top 10 articles that have appeared these last three years. If this is of interest I can do the same for more top articles in another blog.

1. Pamoates and Embonate Salts

2. Preparation of Pharmaceutical Salts

3. Recycling Mother Liquors in Chemical Process Development to Raise Yields

4. Solvent Replacement in the Plant at Scale

5. Getting Better Recovery from Recrystallization

6. Claisen’s Alkali Reagent for separating very Weak Acids like Enols and Cryptophenols

7. The Problem of Oiling Out in chemical Process Development

8. What Might be the Best Cleaning Solvent for Cleaning the Reactor Walls of a Plant Reactor

9. The 1,2-Diol Functionality as a Phase Tag for Process Separation

10. 10. PEG 400 (polyethylene glycol liquid) as a Useful Organic Reaction     Solvent

How does one Produce the Most Likely Impurities in the Product from a Process Step?

In order to develop a good purity analysis for an organic substance, one needs to have some method to assess different methods. A better method separates and quantifies more impurities from the product. A better method increases the degree of separation between the closest impurity and the product without losing separation for any other impurity. A better method separates more cleanly an impurity designed to have a very minor structural difference from the product.

 

But is there a way to prepare a  resolution standard with larger amounts of the most likely potential impurities of the product? This becomes an important practical matter.

There are two types of impurities. Impurities that are derived from product degradation; that is, they come via the desired product and arise from reactions of the desired product occurring after it has been isolated and purified. A separate type of impurity is one that is formed at the time of the synthesis of the desired product and which is not completely removed by the isolation, separation, and purification processes performed before packaging the final product. Such impurities are characteristic of the process. 

It is this second type that is considered here. These impurities are produced in greater or lesser amounts by variations from the proper continuous variables controlling the process.

 

New previously unobserved impurities are usually created by changing the discontinuous variables of the process step, such as reagents, reagent purity, reagent/substrate ratio, solvent, solvent purity, substrate purity, processing chemicals…

The most significant continuous variables are time and temperature.

The one that can produce the most profound or substantial changes in chemical reactivity is temperature. The effect of time is usually more limited because if we are seeking a complete reaction it is likely that the transformation is self-terminating and will essentially stop when the correct time has expired with relatively less occurring after this required reaction time. Increasing the temperature by 10 C according to a rule of thumb should double the rate of reaction. This will also allow competing reactions that are limited under the most preferred conditions to compete and produce by-products.

Thus, an increase in the temperatures of each of the different stages of the reaction by 10C and a decrease in the time by half in each stage should produce more impurities in the final product and these impurities should reflect realistic possible impurities.   If possible the extent of disappearance of starting material should be kept about the same.

Product separated from a process step stressed in this way should show increased amounts of the most likely process impurities.

If it does not, this is important information for your determination of critical parameters that you will need to work on at some point. Tests that provide a resolution standard go hand-in-hand with examining critical parameters!

Uncommon Solvent Immiscibilities

 KiloMentor is always on the lookout for methods to separate components of a mixture by partitioning between immiscible liquids. Better known ones are methanol or acetonitrile with hydrocarbons. Many different solvent pairs may show immiscibility between ambient temperature and -20 C and this temperature range is easily accessible inside a jacketed reactor where liquid-liquid partitioning is done at scale. It is in the laboratory that this temperature range is inconvenient to achieve.

Below are listed some less-common immiscible pairs that may prove useful.

Dimethylsulfoxide - Xylene

Dimethylsulfoxide - Diethyl Ether

Dimethylformamide - Xylene

Dimethylformamide -Diisopropylether

Trichloroethylene - Xylene

Acetic acid - Hexane

Methyl t-Butyl Ether (TBME) -Sulfolane

The DMF /Diisopropyl ether immiscibility suggests that one look for an Upper Critical SolutionTemperature (UCST) between DMF and TBME at below room temperature. A small amount of water could be added to the DMF to raise the UCST.

Since DMSO and diethyl ether have immiscibility it suggests that one explore for a UCST between DMSO and TBME below ambient temperature.

What would the miscibility be between a mixture of xylene and diethyl ether with DMSO? Both xylene and diethyl ether are separately immiscible with DMSO.

To get rid of the diethyl ether suppose we try a mixture of xylene and TBME with DMSO?

How about a mixture of DMSO and trichloroethylene with xylene? Both DMSO and trichloroethylene are separately immiscible with xylene.

Dimethylsulfoxide or DMF reactions could be worked up by extraction into m-Xylene followed by azeotropically removing the xylene as an azeotrope with water after cold extracting the xylene to remove residual dipolar aprotic solvent.

Both m-xylene and isopropylbenzene form azeotropes with water that can be used to quickly remove the organic as a clean phase. Can either of these be useful for isolating organics formed in the solvents DMF or DMSO? You tell me- I’m retired; you have a lab.

Diphenylphosphine Oxide Containing Compounds: Intermediates almost guaranteed to be Crystalline

 

 Stuart Warren, in an article in Accounts of Chemical Research 11 (11) 401 (1978), wrote that almost all diphenylphosphine oxide-containing compounds are highly crystalline white solids. KiloMentor is, therefore, proposing the use of compounds containing the diphenylphosphine oxide substructure as one of the preferred intermediate types in ‘paper’ syntheses.

It is well known that the reaction of a primary alkyl halide with triphenylphosphine produces a quaternary phosphonium salt that is both an ionic salt and crystalline. Hydrolysis of such a compound in aqueous base liberates benzene and provides the phosphine oxides. These compounds in turn can be alkylated with other alkyl halides using butyllithium and TMEDA as co-solvent. [J. Chem. Soc. Perkin Trans. I, 550 (1977)] Warren predicts that these also will be highly crystalline solids.

The KiloMentor strategy for paper synthesis route design emphasizes the advantages of selecting a route that can easily be scaled up. To be preferred, intermediates need to have an increased likelihood of being easily separated and purified, preferably by acid-base extraction. This is proposed to be an overarching advantage over competing routes, whose intermediates almost always have to be purified by crystallization. The problem with these competing routes is that the crystallizability of an intermediate from a paper synthesis cannot be dependably predicted.  

Besides those intermediates, purifiable by extraction, other intermediates would also be preferred if, even when still unknown and existing only ‘on paper’, they contained a functional group that could pretty well guarantee they would be found to be crystalline. There are not many of these and they are not celebrated for this property. Usually, the ease of crystallization for a compound depends upon the entire molecular structure and cannot be predicted, but diphenylphosphine oxide appears to be one that should come with a guarantee.