What's That in My Protein? Degraded Polysorbate Again?

By Dr. Sheri Glaub

Mahler, et. al. have recently published a paper in Pharmaceutical Research entitled, “The Degradation of Polysorbates 20 and 80 and its Potential Impact on the Stability of Biotherapeutics.” (Subscription required.) As discussed in the paper, polysorbates are the most widely used non-ionic surfactants for stabilizing protein pharmaceuticals against interface-induced aggregation and surface adsorption.

Unknown Blogger uses a beater to induce aggregation in a protein solution

Concerns with polysorbate lot-to-lot variability, as well as potential degradation products prompted the authors to investigate the impact on four different monoclonal antibodies (mAbs).  They performed an extensive characterization of polysorbate degradation products, both volatile and insoluble, which included a number of ketones, aldehydes, furanones, fatty acids, and fatty acid esters. They then examined the effect of degraded PS on these proteins.

They concluded that as long as threshold levels of PS20 and PS80 were present (in this case >0.01%), the stability of the four mAbs in pharmaceutically relevant storage conditions (2-8 °C) was maintained despite observed polysorbate degradation.

The authors also suggest during formulation development one evaluate carefully the amount of PS to be used, considering the shelf life and potential behavior during storage.

Getting a grip on prophage

by Dr. Ray Nims

In a previous post, we discussed bacteriophage as a risk for the manufacture of biopharmaceuticals by bacterial fermentation. We mentioned briefly that bacteriophage may integrate within the genome of bacterial cells and that this may also represent a problem. Now we will explain why.

Bacteriophage are viruses that infect bacteria, and they have evolved two mutually exclusive strategies for survival. One involves a lytic growth cycle leading to death (lysis) of the host cell and release of progeny phage that may then infect additional host cells (so-called horizontal transmission). The other strategy is called lysogeny and involves integration of phage coding sequences into the host (bacterial) cell genome. The integrated phage is termed a prophage. This strategy for phage survival is referred to as vertical transmission since the phage genomic material is reproduced along with that of the host cell as the latter proliferates. Under certain circumstances, however, the integrated prophage can excise itself from the host cell chromosome in a process referred to as induction. The excised phage then may initiate a lytic infection of the host cell, causing all of the problems discussed in the previous post.

Illustration of a T4 phage infecting E. coli by Jonathan Heras

The relative success (i.e., from the perspective of the phage!) of the lytic vs. lysogenic survival strategies changes with the probability of host cell survival. Lysogeny appears to be a strategy that allows phage to persist during periods of low host cell availability or poor environmental (e.g., nutrient) conditions. Induction of prophage is an adaptation of the phage to host cell damage. This damage usually takes the form of a major stress to the host cell.

If stess can lead to prophage induction, the worry then becomes that some manipulation of a bacterial production cell during biopharmaceutical manufacture could lead to induction and initiation of a lytic phage infection. How can we assess and mitigate the potential for this to occur? There are two approaches: first, we can perform chemical or physical induction studies to determine the likelihood of encountering a prophage in a given production cell; and second, we can engineer the conditions of bacterial growth such that induction of a prophage is discouraged.

Phage induction studies may be performed on the bacterial production cell following initial engineering of the cell or during characterization of the cell bank. The inducing agent most often employed is mitomycin C. Other types of inducing agents (conditions) include carcinogens (such as the N-nitrosamines), hydrogen peroxide, high temperature, starvation, and UV radiation. The cells are treated with the inducing agent or condition, then one of various endpoints is used to detect the initiation of a lytic phage infection. These could include culture assays as well as molecular techniques such as PCR, microarray, or DNA chips.

Suppose you have an E. coli production cell harboring a problematic prophage. What can be done to discourage phage induction? Certain growth procedures have been shown to reduce spontaneous phage induction in E. coli cultures. These include using lower bacterial growth rates, replacement of glucose in growth medium with glycerol, and engineering the production cell through introduction of a plasmid conferring over-expression of the phage cI gene.

In summary, there are approaches that can identify the likelihood of encountering prophage induction from a bacterial production cell. The time to perform this type of testing is during development of the fermentation process (following the engineering of the production cell), or following banking of the production cell. If prophage induction appears to be a problem, bacterial growth procedures can help to reduce the potential. If this is not sufficient, the production cell may need to be re-engineered to produce a phage-resistant mutant.

Cold Facts About Dissolved Oxygen

By Dr. Scott Rudge

What’s the solubility of oxygen in water? Everyone knows that the answer to this question is “low”, and that’s enough to know for many practical applications. But it’s high enough to rust unprotected metal surfaces, and high enough to grow cells, provided that it’s replenished at some rate. It’s easy to find a number on the internet, at a temperature and pressure that the author of the internet resource thinks is interesting. But my interesting condition always varies from the internet’s, and finding the constants that I need to calculate the actual value is always difficult.


Most references that you can find say that the solubility of oxygen in water follows Henry’s Law. Henry’s Law is a very simple expression that says the concentration of a substance in a liquid phase is related to the partial pressure of that substance in the gas phase by a “constant”. Constant is a relative term in thermodynamics, because the constant in this and most cases varies with temperature and other components in both phases. But we’ll go with it.

In this equation, k_H is Henry’s constant, p is the partial pressure of the substance in the gas phase and c is the corresponding concentration of that substance in the liquid phase. Partial pressure is simply the amount of the total pressure due to that substance. In a room full of air at sea level, the partial pressure of oxygen is approximately 21% of the atmospheric pressure, or 0.21 atmospheres (159.6 torr or mm Hg, or 3.1 psi). At 25°C, Henry’s constant for oxygen in pure water is 769.2 L*atm/mol.

To correct for temperature, an approximation using a reference temperature can be used, although the results will not be exact. The correlation is:

Where T_r is the reference temperature. Again, the use of these equations will give approximate values.

Adding salt to the water further decreases the solubility of oxygen, such that in sea water the solubility of oxygen is about 80% of that in pure water. In fermentation medium, the saturation concentration is probably even less.

For those who don’t like math, here’s the table for the dissolved oxygen concentration under air atmospheres containing 21% oxygen (dissolved oxygen concentrations in moles/L):

Lesson learned: Outsource but remain in control!

By Dr. Ray Nims

In a previous posting, we described the responsibilities of the contract giver (contractee) and the contract acceptor (contractor) in outsourced pharmaceutical quality control testing. Our blog title: "Outsource it, and fuggedaboutit?" somewhat facetiously suggested that the outsourcing of quality control testing does not transfer quality control responsibility from the contract giver to the contract acceptor.

Elizabeth Meyers and I expanded upon this theme in a recent article in BioProcess International. Our conclusion in that article was more direct: “The use, by a pharma organization, of a contract testing lab to fulfill some or all of its Quality Control testing obligations does not absolve the contractee of its overall Quality responsibility of ensuring the safety, purity, identity, efficacy, and potency of its products.”

This point was illustrated nicely in a recent warning letter published on the FDA Website. The name of the firm involved is not important to this discussion. Among the other findings was the following:

“Your firm failed to properly evaluate a contract laboratory to ensure GMP compliance of operations occurring at the contract site.”


The FDA then provided the following detail: "...we are concerned about your firm’s fundamental understanding of what is required by your Quality Unit and the regulatory expectations for a firm that enters into agreements with contract testing laboratories. Although you have agreements with other firms that may delineate specific responsibilities to each party, you are ultimately responsible for the quality of your products and the reliability of test results. Regardless of who tests your products or the agreements in place, you are required to manufacture these products in accordance with section 501(a)(2)(B) of the Act to assure their identity, strength, quality, purity, and safety."

The take-home message from this is that in the outsourcing of quality control testing, responsibility for the outsourced testing is retained by the contract giver. Responsibilities of the contract giver include the following: selecting and qualifying the contract lab, ensuring the suitability of methods used (through qualification, transfer, verification, or validation); putting in place a Quality and business agreement; scheduling and submitting samples (including communicating expectations for sample results); providing in-life guidance; and monitoring of contract lab performance.

There is no denying that fulfilling these responsibilities requires a significant and ongoing effort on the part of the contract giver. In this respect, outsourcing of quality control testing is not so different from doing that testing in-house.

What's up with USP Chapter 1050?

by Dr. Ray Nims

United States Pharmacopeia (USP) general chapter <1050> Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin originally appeared in supplement 10 to USP23-NF18 in May 1999 and was at that time essentially a verbatim adoption of the International Conference of Harmonization (ICH) Guideline Q5A (R1) having the same title.

The chapter describes the methods of evaluating the viral safety of biotechnology pharmaceutical products that are manufactured using cell lines of human or animal origin.

In 2006, an ad hoc advisory panel was assembled by the USP and tasked with revision of this chapter. The goals were to update the chapter and, more specifically, to add greater detail in the viral clearance validation section. The hope was that a user following the recommendations set forth in the general chapter would have greater confidence that viral clearance validation data generated would prove acceptable to the regulatory agencies.

The organization of the revised chapter <1050> was not changed. It comprised the following main sections: 1) Introduction; 2)Potential sources of viral contamination; 3) Cell line qualification: testing for viruses; 4) Testing for viruses in unprocessed bulk; 5) Rationale and action plan for viral clearance studies and viruses tests on purified bulk; and 6) Evaluation and characterization of viral clearance procedures. The changes proposed for the initial 5 sections were minor and primarily reflected attempts to update the chapter and to align the chapter more closely with FDA guidance documents. The most extensive changes were to section 6, in keeping with the goals described above.

The revised chapter was published for public comment in Pharmacopeial Forum 36(3) in the fall of 2010. Comments received as a result of the public review apparently suggested that a more extensive update of the chapter was warranted. At any rate, the revised chapter was not made effective during the USP’s 2005-2010 revision cycle. A new ad hoc advisory panel now being assembled as part of USP's 2010-2015 revision cycle will take over the responsibility for moving the revision of this chapter forward.