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.

Got animal-derived materials? Part 2

By Ray Nims

As part of a formal animal-derived materials program, biopharma companies must assess the viral and TSE risk associated with materials derived from animals or which have been in contact with animal-derived materials at some point. We have addressed the viral risk within a previous blog installment. Now let’s consider the TSE risk.

TSEs (transmissible spongiform encephalopathies) are fatal diseases believed to result from exposure of a few animal species (including various ruminants, cervids, ungulates, cats, minks, and humans) to “infectious” prion proteins. The infectious proteins (PrP^Sc) are capable of interacting with and altering the normal prion proteins (PrP^c) within the brain and spinal cord, changing the normal proteins to the abnormal form. Since humans have contracted prion disease as a result of consuming tissues from cattle with bovine spongiform encephalopathy (mad cow disease), there is concern about the use of bovine materials and materials from other “relevant species” in the manufacture of biopharmaceuticals.

The EMEA has provided a guidance document (EMEA/410/01 Rev. 2 October 2003; http://www.emea.europa.eu/pdfs/human/bwp/TSE%20NFG%20410-rev2.pdf) describing the requirements for the use of animal-derived materials from relevant animal species (cattle, sheep, goats, and other animals which are naturally susceptible to TSEs but not including humans or non-human primates) in the manufacture of medicinal or veterinary products. The guidance applies to active substances, excipients and adjuvants, raw and starting materials and reagents, and materials which come into contact with products or equipment used to make product. The major point of the guidance is that TSE risk can be minimized, but in many cases not entirely eliminated. Where TSE risk cannot be avoided through elimination of animal-derived materials completely, the guidance provides principles for the minimization of TSE risk which include: the use of low-risk (non-relevant) animal species, the geographical sourcing of relevant species from low-risk regions, the use of low-risk tissues, the use of appropriate slaughter techniques to reduce potential for contamination of low-risk tissues with high-risk tissues, the appropriate oversight of manufacture of the animal products through Quality Assurance and self and external auditing and Quality Control testing, and the implementation of process designs to remove or inactivate the abnormal prion proteins.

Biopharma companies using animal-derived materials are instructed to perform risk assessments on those materials, which take into account the factors described above. The risk assessments must be completed as part of a formalized animal-derived materials program, documented procedurally and executed by staff that are experienced and trained to conduct such assessments. EMEA inspectors will expect to see this formal program in place. Risk assessments for individual raw materials may then be rolled up into a risk assessment for the biopharma product. The rolled up risk assessment may also consider manufacturing steps at the biopharma which may remove or inactivate the abnormal prion proteins, though these, if cited, may need to be validated. Finally, it is expected that companies will conduct a benefit/risk evaluation to assure that any benefits realized by the patient taking the product will outweigh and justify the risk associated with the use of materials derived from relevant animal species.

Got Animal Derived Materials?

By Ray Nims

Most biopharma manufacturing processes utilize a few raw materials (including cell substrates, excipients, materials which come into contact with the product, etc.) derived from animals or which have been in contact with animal-derived materials at some point. As part of a formal animal-derived materials program, the biopharma must assess the viral and TSE risk associated with such materials. Let’s consider the viral risk first. From a viral safety standpoint, it is important for each biopharma to consider such ingredients and to be aware of the inherent risk of transmitting virus into the product via the materials. Why? As Genzyme discovered in the spring of 2009 (http://www.genzyme.com/corp/media/GENZ%20PR-061609.asp), viruses can infect the upstream manufacturing processes with results devastating to both the biopharma and to the patients its products are intended to treat. Viral risk mitigation, and regulatory guidance (e.g., EP Chapter 5.1.7: Viral Safety), require that viral risk assessments be performed for animal-derived materials used to manufacture biologics. In this context, raw materials include also excipients, growth media, column packing resins, and cell substrates.


For each product, manufacturers should list the animal-derived materials utilized, and should perform a viral risk assessment for those materials. This assessment considers the animal species and tissue, the processes used to manufacture the raw material, the quality control testing performed on the raw material, and in some cases, the manufacturing process in which the raw material is to be used at the biopharma. Inspectors from the EMEA will not only expect the risk assessments to have been performed and documented, but will expect that the assessment process be formalized into a business practice or standard operating procedure. The staff performing the assessments should be qualified for this task and the assessment team should include staff knowledgeable in virology, viral inactivation and removal, and the manufacturing and purification processes employed for the specific product at the biopharma.

Viral risk assessments completed for individual raw materials may eventually be rolled up into a viral safety assessment for the product per EP Chapter 5.1.7. This product evaluation will also consider other factors, such as the patient profile and route of administration, the cell substrate, the types and pathogenicities of viral contaminants found in the cell substrate and the manufacturing process, the amount of bulk material required for a human dose, and the viral inactivation and removal capabilities of the manufacturing downstream processes.

Outsource it, and fuggedaboutit?

By Ray Nims

Much has been written about the rationales and advantages for outsourcing of manufacturing and/or testing services; about the selection of outsourcing partners; and about the optimization of the pharma/contractor relationship. In any pharma/contractor relationship, there are responsibilities associated with the pharma as well as contractor responsibilities. These include both business as well as compliance responsibilities. The business realities and regulatory expectations associated with the use, by a pharma company, of a contract testing organization must be considered when the decision is made to outsource. A contract testing organization desiring to provide services for a pharmaceutical must be aware of the expectations and responsibilities associated with such a partnership. The optimal and most defensible programs will be those in which the various practices to be described below are formalized within internal Quality Systems, policies, and/or standard operating procedures as well as Quality Agreements.


Responsibilities falling upon the pharmaceutical partner include: 1) the selection of the contract testing lab; 2) commissioning and providing test samples of raw materials and products for method verification (compendial methods) and method qualification (non-compendial methods); 3) instituting of a Quality Agreements, business agreement, and/or confidentiality agreement with the contractor; 4) scheduling and shipping of test samples in accordance with the requirements of the testing lab and the test system; 5) providing in-life guidance and oversight of investigations of unexpected and out of specification results; and 6) ongoing monitoring of the performance of the contract lab and its methods.

Responsibilities primarily falling upon the testing lab include: 1) attaining and maintaining GLP or GMP compliance as appropriate for the intended use of the method; 2) providing assurance that the methods offered will be available to the client over the long term; 3) responsiveness to the sponsoring pharma and adherence to the terms of the Quality and/or business agreements; 4) method validation, verification, and or qualification as appropriate for the intended use of the method; 5) control of reagent, raw material, control, and standard inventory and quality; 6) assuring secure and retrievable data archiving; and 7) retention of staff possessing the appropriate expertise for direction of operators and the methods.