How Do You Set Endotoxin Specs on API?

By Dr. Lori Nixon

For parenteral drug products, setting a specification limit for endotoxin is a relatively straightforward exercise. If you know the maximum hourly dose and route of administration, the endotoxin limit can be readily derived from the equations set out in USP<85>.


But how do you determine an appropriate limit for your API? Don’t make the mistake of applying the same limit as product (in terms of EU/mg of active). And don’t make the mistake of blindly applying the same limit as a similar API, that may be dosed differently. Since the drug product is typically a combination of active ingredient along with excipients, solvents and container components—each of which may potentially contribute to the total endotoxin level—you should account for other potential contributions and set your drug substance limits accordingly. By setting the appropriate limit in API, you avoid the risk of an endotoxin failure that doesn’t show up until your precious API has been formulated and filled into vials—a business catastrophe!

Consider the following example:

Drug product at 50 mg/mL, intended for iv delivery in adults.

For this product, the maximum human dose/hour is 250 mg, delivered as a 5 mL volume.

From USP<85>, the endotoxin limit for iv delivery is 5 EU/kg. For a 70 kg adult, this corresponds to 350 EU maximum, or 70 EU/mL.

Tabulate the potential endotoxin contributors from your drug product formulation, container and manufacturing process. Look up the endotoxin limits (specifications) for each component you listed. In some cases it’s not possible to get a hard number, but you can at least estimate or list a “worst-case”. Some components will have negligible contribution based on their low ratio of surface contact to total formulated volume (such as the needle/tubing in the fill line); or low overall volume contribution (such as an acid/base used in a titration step). In this scenario, the sum of endotoxin contributions from all components (including API) cannot exceed 350 EU. “Solve” for the allowable endotoxin contributions from the API (X, in the table).

*Notice that in the table, drug substance is shown as 60 mg/mL (as powder) even though the formulation strength was listed as 50 mg/mL. Recognize that endotoxin measurements are usually based on powder weight rather than active ingredient weight. In this example let’s assume that the API has a moisture specification of NLT 95% and purity specification of NLT 88%; so 50 mg/mL of active (in the worst-case) actually corresponds to 60 mg/mL of API powder. So a 5 mL dose may contain as much as 300 mg of API powder.


You won’t be able to nail down everything exactly, and should recognize that relying on release specifications for some of the components above will clearly result in overestimations for endotoxin contribution. For example, the specification limit for the vials may not take into account additional reduction from depyrogenation; filters would be pre-rinsed and any subsequent endotoxin contributions would be largely diluted within the total product volume; and so forth. Nonetheless, this can be a convenient and systematic way to start your evaluation from a “worst-case” standpoint.

The allowed endotoxin contribution from the API can be calculated by subtracting the sum of the other component contributions (far right column of the table) from the total of 350 EU. In this case, X = 235.7 EU. Divide by the largest amount of drug substance powder that could go into the maximum dose (in this case 300 mg), and you have the drug substance endotoxin limit (0.79 EU/mg).

Now, do a reality check: If this drug substance limit looks like it may be problematic for any reason (analytical or process capabilities), look again at the table to see other possible ways to reduce the overall endotoxin (or revisit some of the worst-case assumptions that were clearly overly exaggerated). In the example, Salt 2 appears to be a large potential contributor, with a fairly loose specification for endotoxin. It may be possible to specify a different grade of this excipient (low-endotoxin or parenteral grade). Remember that compendial grades don’t necessarily mean low-endotoxin grades or even endotoxin-controlled grades.

Innovators and Perfectionists

by Dr. Ray Nims

In a previous posting, Leah Choi described her frustration over the lack of specific training, received during her undergraduate schooling, in aspects germane to the realities of employment within the biopharmaceutical industry. Employment in the highly regulated world of biopharmaceutical manufacturing, Quality, and Quality Control (biopharmaceutical operations) requires different skills and employee temperaments compared to employment within the research and development (R&D) world. The academic institutions would do well to consider this during the preparation of students for eventual life in the working world.

What do we mean by different skills and employee temperaments? Most of us are familiar with the Myers and Briggs personality typing instrument which looks at attibutes like intro/extraversion, sensing, intuition, etc. This instrument provides interesting and revealing information about how different people deal with the world. In addition to the qualities addressed by Myers & Brigg, however, there is a personality spectrum which I will refer to as Innovation ↔ Perfection.

In the R&D world, technical knowledge, mastery of the literature concerning a subject, and most importantly, innovation are the attributes which are essential for success. A researcher must long to travel untraveled paths, to uncover new ground, to learn and often develop methods where such may not have existed previously (i.e., to go where no man has gone before). Those who are well suited to this environment we refer to as innovators. The academic institutions are pretty good at fostering these attributes in students.

On the other hand, the highly regulated areas of biopharmaceutical operations require individuals who are capable of following set instructions time after time, documenting their work in a precise and strictly controlled manner. Innovation, improvisation, and experimentation with mature methodologies and standard operating procedures are not encouraged. A mind-set which is compatible with achieving perfect compliance with documented procedures is the key to success in this environment. Such individuals we refer to as perfectionists, as they are motivated by the desire (or if not desire, at least the requirement) to conduct their work exactly as proscribed. It is this particular set of attributes which many academic programs fail to address adequately. This leaves employers with the task of training their entry-level staff in such matters, and (as Leah mentioned in her posting) students with the sometimes shocking revelation that they are poorly prepared for this type of employment.

Are there individuals who can be successful both as “innovators” and as “perfectionists”? Undoubtedly so! It is more likely, however, that most people fit within a spectrum falling between the two temperaments. I, for instance, have always regarded myself more of a perfectionist than an innovator, happily conducting the same assay the 100th time and still trying to do a better job than the last time. I know of others who, as soon as they learn a method, are bored with it and anxious to move on to something new. These temperaments may be determined by our personalities and may not be subject to alteration. It would appear to be valuable for academic programs to try, therefore, to determine the temperaments of their students, and to provide training suitable and appropriate for both the innovators and the perfectionists. Both the students as well as the biomedical industry would benefit from a little temperament triage and curriculum adjustment done at the undergraduate level.

Assessing rapid mycoplasma detection systems

by Dr. Ray Nims

The European Pharmacopoeia chapter 2.6.7 Mycoplasmas, begining with version 5.8, has provided a mechanism for replacement of the current 28-day culture method for detection of mollicute (mycoplasma and acholeplasma) contaminants in biopharmaceutical bulk harvest samples with more rapid, nucleic acid-based, methods. The US FDA has yet to provide formal guidance on this topic, although it has become clear that the agency is willing to consider such methods, provided that they are shown to be equivalent to or superior to the current approved methods.

For biopharmaceuticals, a satisfactory outcome in a mycoplasma detection assay which is compliant with European Pharmacopoeia 2.6.7 or the 1993 FDA Points to Consider guidance is required on a lot-by-lot basis. Of the various lot-release assays performed on each given lot of a biopharmaceutical, this particular test is typically the most lengthy. Expediting the lot-release process through replacement of the 28-day approved culture test with a rapid mycoplasma detection test is therefore a strong motivating factor for the biopharmaceutical industry.

Figure. The MicroSEQ Mycoplasma assay provides a level of detection less

than 10 CFU/ML.

What options are now available to the industry? Several contract testing laboratories have recently announced the availability of validated rapid mycoplasma assays suitable for biopharmaceutical lot-release. For instance, BioReliance offers a hybrid culture/quantitative polymerase chain reaction (qPCR) assay, Charles River Laboratories offers a reverse transcriptase (RT)-PCR assay (BioProcess Int. April 2009, 30-42), Vitrology offers a qPCR assay, and WuXi AppTec offers a “touchdown” PCR assay.

In addition, several vendors are now offering mycoplasma detection kits which will allow biopharmaceutical entities to perform rapid mycoplasma testing in-house. For example, Life Technologies offers the MicroSEQ® Mycoplasma Detection Assay, Roche Applied Science offers the MycoTool™ PCR test and Millipore offers the MilliPROBE® mycoplasma detection system.


It is incumbent upon the biopharmaceutical company to demonstrate comparability between the rapid mycoplasma method and the current approved culture method for each product matrix for which a rapid method is proposed. Guidance on such comparability testing is provided in the European Pharmacopoeia chapter 2.6.7. Comparability studies for rapid methods intended to satisfy the US FDA should be discussed with that agency, as no formal guidance has been published.

What attributes should be considered when selecting a rapid mycoplasma detection method?

1. Sample volume. The current approved culture methods test at least 10 mL of sample. A rapid method intended to replace the current methods should ideally be able to test an equivalent volume of sample. It may be difficult to gain FDA approval for nucleic acid-based methods which can test only microliter amounts of sample.
2. Duration. Hybrid culture/PCR systems may take as long as 14 days to complete, while direct nucleic acid-based methods should be completed within a week or less.
3. Specificity. European Pharmacopoeia 2.6.7 specifies that the nucleic acid test must be able to exclude closely-related bacterial species.
4. Sensitivity. FDA indicates that the rapid method should be equivalent to or better than the approved culture method in terms of sensitivity (limit of detection), based on comparability studies using viable mycoplasma organisms.
5. Orthogonal endpoints. Having two or more orthogonal endpoints is desirable to allow one to discriminate between low level positive and negative signals.
6. Validation status. For contract methods, has the method been validated per European Pharmacopoeia 2.6.7? For kit methods, has the vendor validated the method per European Pharmacopoeia 2.6.7?
7. Drug Master File. For kit methods, has the vendor submitted a drug master file to the FDA for the method?

These considerations should help in deciding among the various options now available for implementing rapid nucleic acid-based mycoplasma testing for biopharmaceutical lot release applications.

QbDer, Know Thy Model!

By Dr. Scott Rudge

Resolution in chromatography is critical, from analytical applications to large scale process chromatography. While baseline resolution is the gold standard in analytical chromatography, it is seldom achieved in process chromatography, where “samples” are concentrated and “sample volumes” represent a large fraction of the column bed volume, if not multiples of the bed volume. How do you know if your resolution is changing in process chromatography if you can’t detect changes from examining the chromatogram?


Many use the Height Equivalent to a Theoretical Plate technique to test the column’s resolution. In this technique, a small pulse of a non-offensive but easily detected material is injected onto the column, and the characteristics of the resulting peak are measured. The following equation is used:

Where H is the “height” in distance, t_r and t_w,1/2 are the retention time and time of the width of the peak at ½ height, L is the length of the column and N is the “number” of theoretical plates in the column. The lower the H, the smaller the dispersion, the greater the resolution.

It is well established that the flowrate and temperature affect the plate height. In fact, when the plate height is plotted against flow rate, we generate what is typically called the “van Deemter plot”, after Dutch scientists (van Deemter et al., Chem. Eng. Sci.,5, 271 (1956)) who established a common relationship in all chromatography (gas and liquid) according to the following equation:

Where A, B and C are constants and v is the linear velocity (flow rate divided by the column cross sectional area) of fluid in the column. It was later proposed by Knox (Knox, J., and H. Scott, J. Chromatog., 282, 297 (1983)) that van Deemter plots could be reduced to a common line for all chromatography if the plate height was normalized to resin particle size, and linear velocity was normalize to the resin particle size divided by the chemical’s diffusivity. While this did not turn out to be generally true, it is very close to true. Chemical engineers will recognize the ratio of linear velocity to diffusivity over particle size as the Peclet number, “Pe”, a standard dimensionless number used in many mass transfer analyses.

Since diffusivity is sensitive to temperature, it is logical that Pe is also sensitive to temperature, decreasing temperature decreases the diffusivity, and increases Pe. Thus, Pe is inversely proportional to temperature. We measured plate height as a function of linear flowrate and temperature in our lab on a Q-Sepharose FF column, using a sodium chloride pulse, and found the expected result, shown in the graph below.

We can easily use this graph as a measurement of van Deemter’s parameters A and C, and find the dependence of the diffusivity of sodium chloride on temperature. Based on these two points, we find the Peclet number proportional to 0.085/T where T is in degrees Kelvin. We also find the dependence of plate height on linear velocity, and we can predict that resolution will deteriorate in the column as flow rate increases.

We can also use Design of Experiments to find the same information. Analyzing the same data set with ANOVA yields significant factors for both flow rate and temperature, as shown in the following table:

Term	Coef	SE Coef	T	P
Constant	0.09756	0.05604	1.74	0.157
Temp	-0.007935	0.001858	-4.27	0.013
v	0.0497	0.02147	2.32	0.082

But since the statistics don’t know that the temperature dependence is inverse, or that the Peclet number is a function of both temperature and flowrate, the model yields no additional understanding of the process.

It is possible to use analysis of variance to fit a model other than linear, as the van Deemter model clearly is. But one must know that the phenomenon being measured behaves non-linearly in order to use the appropriate statistics. Using Design of Experiments blindly, without knowing the relationship of the factors and responses, leads to empirical correlations only relevant to the system studied, and should only be extended with caution.