Got Animal-Derived Materials? Part 3

By Dr. Ray Nims

The assessment of viral and transmissible spongiform encephalopathy (TSE) risk for animal-derived materials (ADM) used in the manufacture of biologics, which we have described in previous blogs, is just one component of an overall ADM program that should be in place at each organization producing biologics.

A formal ADM program at a biologics manufacturer ideally should be driven by an overriding SOP or policy document. This should address the procedures in place for minimizing the use of ADM, for procuring ADM with a view to minimizing viral and TSE risk, and for assessing the viral and TSE risk associated with the ADM that are used. There are specific sourcing requirements for ADM that are intended to minimize TSE risk (EMEA/410/01 Rev. 2 October 2003), and these must be followed or justification provided if deviated from. The evaluation of ADM for the presence of viruses of concern is addressed in the Code of Federal Regulations, Title 9 Part 113.53. ADM viral and TSE risk assessments should be conducted according to a formalized procedure by teams of individuals with education, training, and/or experience appropriate for these tasks. The composition of the risk assessment teams and the qualifications of their members should be described in revisable controlled documents. The risk assessments themselves should be recorded in controlled documents which may revised as new information becomes available from the ADM suppliers. The ADM information that is used as part of the risk assessment process should be archived in a manner tying it to the risk assessment itself.

The existence of a formalized ADM program, qualified risk assessment teams, as well as reports documenting the individual ADM risk assessments may be the subject of regulatory scrutiny during periodic inspections or inspections tied to a new product application. This is especially likely if the product is intended for global distribution, as these ADM issues are specifically mentioned in EP (Chapter 5.1.7) and EMEA (EMEA/410/01 Rev. 2 October 2003) guidance.

Advantages of Compendial Methods

By Dr. Lori Nixon

When you are developing a new product specification, it is usually recommended to rely on the appropriate compendial method for applicable “generic” quality characteristics such as pH, residual solvents, trace metals, bioburden, etc. By compendial method, we mean methods that are described as chapters in the United States Pharmacopeia (USP) or others that may be applicable for a specific regulatory region. The three main compendia include the USP, European Pharmacopoeia, Japanese Pharmacopeia (USP, PhEur, JP); these are the “tripartite” bodies that are involved in the International Conference on Harmonization (ICH).

photo of USP laboratories from DPR Construction Inc.

Why rely on compendial methods rather than just using your own? It is generally recommended to refer to compendial methods where applicable. The advantage to the drug sponsor is a reduced requirement for validation supporting such methods (the methods themselves are considered validated, and may only require product-specific verification in the particular testing lab). Compendial methods are “familiar” to regulatory reviewers; they are also generally expected. If you propose your own method as an alternate method, you will need to justify why your own method is equivalent or better. For the testing lab, there is some advantage in having methods that can be applied to multiple products (avoiding a multiplicity of similar methods) and where the change process is relatively well-defined and publically communicated. You may also find it simpler to transfer testing between different labs.

To reference the compendial method in your specification, you may refer simply to the test by attribute and chapter, along with the associated limit for your product. For example, your specification may include a limit of 10 EU/mL for bacterial endotoxin as measured by USP<85>. The general expectation here is that at the time of testing, the current version of USP is used. Clearly, this will require that your testing lab is aware of any potential changes to the USP and can prepare for such changes accordingly. As with any other change to an analytical method, changes to compendial methods can impact training, internal procedures, product-specific re-validation/verification, etc.

In practice, labs often rely on additional internal descriptive procedures in order to execute the compendial methods (i.e., rather than just directing analysts to follow the chapter directly). This is usually a good idea, for several reasons. It can be easier to train analysts according to a standard documentation format, and it is often necessary to describe details that may be specific to the particular lab, equipment, instrumentation, reagents, reporting requirements, etc. Again, even if there is a lab-specific procedure, it is usually best to refer directly to the compendial method (USP<85>, e.g.) in the sponsor’s product specification.

Be aware of compliance with compendial testing requirements when you are outsourcing testing. For example, almost any chemical testing lab with have a method for pH, but that doesn’t necessarily mean that it will comply with USP<791>. For example, in this case the USP method describes measuring the sample temperature within a certain range; often “generic” lab methods for pH do not specify control of the sample temperature. There are additional requirements such as the calibration standards chosen, etc that must also be considered. When reviewing vendor methods, check the following:

- Does the method purport to comply with any compendia? (should be clearly stated in the vendor’s procedure if so)

- Which compendia?

- Check details of the method to ensure that it does indeed comply with the current compendial procedure(s) in question

- Does the lab have a mechanism to stay current with upcoming compendial changes?

- Do they have appropriate change control system to ensure that they can prepare for method changes and associated re-validation, etc?

- Consider what verification/validation is required to ensure that the vendor method provides reliable results for your particular product/sample type.

Of course, the downside of compendial methods is that they are region-specific, and one region may not recognize the compendia of another region. There have been efforts in recent years towards harmonizing methods (ICHQ4B for bioburden testing, for example), but this process is slow and far from complete.

If you intend to market or perform clinical trials in more than one region, you may need to ensure compliance with multiple compendia. In this case, consider the following:

• Has method been harmonized through the ICH process?

• Is it possible to create an internal “harmonized” method that meets the requirements of all relevant compendia?

• Is it possible to meet the requirements of all by following the “most stringent” compendial procedure?

• Will you need to test by multiple procedures to generate results acceptable to each region?

Should we care about…Vesiviruses?

By Ray Nims

Vesiviruses are single-stranded RNA viruses of family calicivirus, genus Vesivirus. They are non-enveloped and 30-40 nm in diameter, and the genus includes feline calicivirus, vesicular exanthema of swine virus, rabbit vesivirus, and San Miguel sea lion virus, as well as vesivirus isolate 2117.

source: Stewart McNulty, Queens University, Belfast, UK

Basis of Concern. Vesivirus 2117 has been isolated from biologics manufacturing processes employing Chinese hamster cell substrates on a number of occasions, the first being reported in 2003 (Oehmig et al., J. Gen. Virol. 84, 2837-2845, 2003), and additional occurrences being reported in 2008 and 2009.
The susceptibility of relevant manufacturing cell lines of different animal species to infection by this virus appears to be limited to the Chinese hamster. When infected, these cells undergo a relatively rapid lytic infection. The route of entry of the virus into biologics production processes has not been established with certainty, although the use of contaminated animal-derived materials, such as bovine sera, is considered to be the most likely source.

Regulatory Expectations. Vesivirus is not mentioned specifically in any regulatory guidance, as the detection of the 2117 isolate in biologics production has been reported only within the past decade. It is the intent of the guidance, however, that occurrences of viral contamination in biologics manufacturing be dealt with through implementation of specific testing methods as required to assure detection of future recurrences (e.g., ICH Q5A R1). In addition, it is expected that the route of entry of the virus be established and that the process be remediated so that future recurrences are prevented where possible (e.g., 1997 Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use).

Mitigating Risk. At least three Contract Testing laboratories have announced rapid nucleic acid-based detection assays for vesivirus isolate 2117 within the past year. These assays are available for raw material screening and for in-process testing of biologics bulk harvest samples. Elimination of animal-derived materials (esp. bovine sera) from the manufacturing process may help to reduce the risk of experiencing this virus. Should this not be possible, treatment of the sera or sera-containing media should be considered. Studies on the inactivation of caliciviruses indicate that UV treatment may be effective (Duizer et al., Appl. Env. Microbiol. 70, 4538-4543, 2004; de Roda Husman et al., Appl. Env. Microbiol. 70, 50989-5093, 2004). Gamma-irradiation at the dosages normally used does not appear to be effective, as might be expected for a virus of this relatively small size. Studies using MMV indicate that high-temperature short-time (HTST)-treatment of medium containing bovine serum is effective in inactivating this virus (Schleh et al., Biotechnol. Prog. 25: 854-860, 2009), and would by implication be effective for vesiviruses in general.

Conclusions. Vesivirus isolate 2117 preferentially infects Chinese hamster cells and has been found to contaminate biologics manufacturing processes employing this cell substrate. It is now a virus of concern for the biopharmaceutical industry. Risk of infection of biological products with vesiviruses through use of bovine-derived materials such as bovine sera may be mitigated through implementation of UV or HTST treatment of media containing the sera and of viral purification processes capable of removing and inactivating an even smaller non-enveloped virus such as MMV.

The Good Buffer

By Scott Rudge

“A Good Buffer” has a number of connotations in biochemistry and biochemical engineering. A “good buffer” would be one that has good buffering capacity at the desired pH. The best buffering capacity is at the pK of the buffer of course, although it seems buffer salts are rarely used at their pK.

Second, a good buffer would be one matched to the application. Or maybe that’s first. For example, the buffering ion in an ion exchange chromatography step should be the same charge as the resin (so as not to bind and take up resin capacity). For example, phosphate ion (negative) is a good choice for cation exchange resins (also negatively charged) like S and CM resins.

Another meaning of a “Good” buffer is a buffer described by Dr. Norman Good and colleagues in 1966 (N. E. Good, G. D. Winget, W. Winter, T. N. Connolly, S. Izawa and R. M. M. Singh (1966). "Hydrogen Ion Buffers for Biological Research". Biochemistry 5 (2): 467–477.). These twelve buffers have pK’s spanning the range 6.15 to 8.35, and are a mixture of organic acids, organic bases and zwitterions (having both an acidic and basic site). All twelve of Good’s buffers have pK’s that are fairly strongly temperature dependent, meaning that, in addition to the temperature correction required for the activity of hydrogen ion, there is an actual shift in pH that is temperature dependent. So, while a buffer can be matched to the desired pH approximately every 0.2 pH units across pH 7 ± 1, the buffers are expensive and not entirely suited to manufacturing applications.

In our view, a good buffer is one that is well understood and is designed for its intended purpose. To be designed for its intended purpose, it should be well matched to provide adequate buffering capacity at the desired pH and desired temperature. As shown in the figure, the buffering capacity of a buffer with a pK of 8 is nearly exhausted below pH 7 and above pH 9.

It’s easy to overshoot the desired pH at these “extremes”, but just such a mismatch between buffering ion and desired pH is often specified. Furthermore, buffers are frequently made by titrating to the desired pH from the pK of the base or the acid. This leads to batch to batch variation in the amount of titrant used, because of overshooting and retracing. In addition, the temperature dependence of the pK is not taken into account when specifying the temperature of the buffer. Tris has a pK of 8.06 at 20°C, so a Tris buffer used at pH 7.0 is already not a good idea at 20°C. The pK of Tris changes by -0.03 pH units for every 1°C in positive temperature change. So if the temperature specified for the pH 7.0 buffer is 5°C, the pK will have shifted to 8.51. Tris has 3% of its buffering capacity available at pH 7.0, 5°C, it’s not well matched at all.

A good buffer will have a known mass transfer rate in water, so that its mixing time can be predicted. Precise amounts of buffering acid or base and cosalt are added to give the exact pH required at the exact temperature specified. This actually reduces our reliance on measurements like pH and conductivity that can be inexact. Good buffers can be made with much more precision than ± 0.2 pH units and ± 10% of nominal conductivity, and when you start to make buffers this way, you will rely more on your balance and understanding of appropriate storage conditions for your raw materials, than making adjustments in the field with titrants, time consuming mixing and guessing whether variation in conductivity is going to upset the process.

Enzyme induction…done pharmacodynamically

By Ray Nims

Pharmacodynamics is the study of a specific effect of a drug as related to drug concentration at the putative active-site for that effect. Pharmacodynamics is sometimes used to model quantitatively the effect of a drug over time as drug concentration at the active-site rises and falls. Another type of pharmacodynamic study entails exposing the animal or in vitro system to graded doses of a drug and monitoring the effect associated with each active-site concentration. From the latter type of study, one is able to estimate both potency for the effect (given in terms of the active-site drug concentration at the half-maximal effect for that drug, or EC50) and its efficacy (given in terms of percentage of maximal response compared to other drugs causing the same effect through the same mechanism). In receptor theory, EC50 is considered to reflect the affinity of the drug for a receptor, while efficacy is a measure of the bound drug’s ability to cause the specific response.


The induction of drug-metabolizing enzymes, such as the cytochromes P450, may be considered to represent an effect of a drug or xenobiotic. It is common for investigators to measure such induction at one or a few dose levels and to compare the resulting enzyme induction with that of a prototype inducer. These comparisons are sometimes described in terms of the test xenobiotic causing “strong” (“potent”) or “weak” induction in comparison with the prototype inducer. As already pointed out quite elegantly by D. A. Smith and coworkers (Letter to the Editor: The Time to Move Cytochrome P450 Induction into Mainstream Pharmacology is Long Overdue. Drug Metab. Dispos. 35:697-698, 2007; http://dmd.aspetjournals.org/cgi/content/full/35/4/697), such statements are both misleading and inaccurate. As with any drug effect, enzyme induction must be described in terms of both potency and efficacy. It is possible for an inducer to be very potent but to display little efficacy. In fact, a xenobiotic having high potency and little or no induction efficacy might represent a competitive inhibitor for this effect. In contrast, there may be inducers which are very effective, but not very potent.

It is possible for efficacy and potency for enzyme induction to be estimated on the basis of studies using intact animals, provided that certain assumptions are made (e.g., that total plasma drug concentration is a suitable proxy for drug concentration at the induction active site, which cannot be sampled directly). An example of such a study is that of R.W. Nims and coworkers (Comparative Pharmacodynamics of Hepatic Cytochrome P450 2B Induction by 5,5-Diphenyl- and 5,5-Diethyl-substituted Barbiturates and Hydantoins in the Male F344/NCr Rat. J. Pharmacol. Exp. Therap. 270: 348-355, 1994; http://jpet.aspetjournals.org/cgi/content/abstract/270/1/348). A more straightforward approach is offered through in vitro enzyme induction studies, in which enzyme induction can be related to drug concentration in the culture medium (e.g., Kocarek and coworkers: Differentiated Induction of Cytochrome P450b/e and P450p mRNAs by Dose of Phenobarbital in Primary Cultures of Adult Rat Hepatocytes. Mol. Pharmacol. 38:440-444, 1990; http://molpharm.aspetjournals.org/cgi/content/abstract/38/4/440).

Measurement of the induction of the cytochromes P450 and other drug-metabolizing enzymes following drug treatment in animals and humans is an important aspect of drug characterization. The studies should be performed and reported in a manner consistent with other drug effects, that is, in a manner consistent with the principles of pharmacology.