Looking beyond your typical day-to-day work and finding out what ‘the rest of the world’ is doing can be a lot of fun. This is one of the reasons I’m enjoying myself so much at the PepTalk 2011 meeting that is currently taking place in San Diego. As I’m learning about the unique challenges faced in protein drug formulations I am quite surprised at the arsenal of analytical techniques that are available to investigate the ‘health’ of a protein prep.
With respect to protein crystallization there has been one presentation by a colleague of Bhami Shenoy from Althea that I found rather inspiring.
The purpose of biologics formulation is to provide a high concentration of pure, stable and functional protein sample in a form that can be kept for an extended period of time, for instance 2 years at room temperature, while showing little decay (no chemical decomposition, aggregation or particle formation). Apparently several protein microcrystaline suspensions are in late stage clinical trials and are an attractive dosage form for several reasons: high protein concentration (>100 mg/ml) solution with low viscosity (patients like their injections to be quick and painless), high stability and slow release of the protein into the bloodstream (lack of burst).
The part of the talk I enjoyed most was a comparison between the requirements of X-ray protein crystallography with that of biologics formulation. Here's a short summary:
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Property
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X-ray crystallography
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biologics formulations
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Crystal size
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>100 um
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0.1-100 um
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Crystal quality
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very important
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not important
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Growth rate
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not important
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important
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Yield
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not important
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very important
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Scalability
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not important
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very important
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Overall, the different requirements are driven by the need for low process costs in biologics formulation. After all, short timelines reduce cost. Curiously, the choice of precipitation is rather limited as compared to what we do for crystal growth for X-ray diffraction experiments, since the crystal suspensions are to be injected into people (better not use that cacodylate buffer system that X-ray crystallographers appear to like so much). The toolset consists of a variety of pH values, a few salts, dicarboxylic acids, sugars, amino acids and detergents. No ammonium sulfate, no polyethylene glycol, no MPD. This makes the identification of productive crystallization conditions rather difficult.
Original data on Infliximab and long acting human growth hormone was presented, showing lowered viscosity (and what was called ‘syringibility’), low/no toxicity or injection site reactions, improved stability and extended release profile while retaining activity and efficacy. I was also impressed by the fact that protein therapeutics crystallizations at production scale are typically carried out in batches of several hundred liters, producing kilo gram amounts of protein crystalline material. I’ll think about that next time I’m setting up a 0.5 + 0.5 microliter vapor diffusion protein crystallization experiment.
Greetings from sunny San Diego,
Peter
Starter for biologics crystallization:
Jen A, & Merkle HP (2001). Diamonds in the rough: protein crystals from a formulation perspective. Pharmaceutical research, 18 (11), 1483-8 PMID: 11758753