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Pharmaceutical Manufacturing and Packing Sourcer

Under Stress

The increasing use of proteins as therapeutics has focused attention on the need to maintain the stability of these labile molecules during both storage and shipment. Chemical damage can occur due to exposure to light and leachables from primary containers, including oxidants, free radicals and metal ions. Adsorption to the surfaces of primary containers, as well as exposure to heat or agitation, can cause proteins to aggregate.

In addition to loss of potency and valuable drug product, there is growing evidence that protein aggregates are capable of inducing an immune response which could neutralise the effect of the drug (1). In instances where the drug product is similar or identical to an endogenous protein, the development of cross-reacting antibodies could lead to potentially life-threatening consequences for the patient.

The trend in the pharmaceutical industry has been to package therapeutic proteins, particularly monoclonal antibodies (mAbs), at high concentration in prefilled syringes. However, vials continue to be used as primary containers for multiple-use applications – including vaccines – as well as for the reconstitution of lyophilised drug products. For this reason, we have compared the effect of mechanical stress on protein aggregation in vials made of glass – the material most widely used in their manufacture – with vials made from the plastic cyclic olefin polymer.


1. To develop and characterise a simple stress model to compare the stability of biologics in vials made of different materials
2. To investigate the aggregation of various classes of proteins, including antibodies, enzymes and peptide hormones in vials made of glass or plastic


The materials used in this comparison consisted of the below:

Pre-sterilised 2ml vials made of glass or the plastic Daikyo Crystal Zenith® (CZ). Stoppers were laminated with FluroTec®, a fluoropolymer film, to minimise the effect of the elastomeric component on protein stability.

Rabbit immunoglobulin G (IgG) was obtained from Rockland Immunochemicals. Therapeutic proteins were purchased from a local pharmacy.

Proteins were dissolved in or diluted into one of two buffers:

  • Phosphate buffered saline (PBS) – 20mm sodium phosphate and 150mm sodium chloride (NaCl) at pH 6.8
  • Phosphate-citrate buffered saline (PCBS) – 20mm sodium phosphate, 2.7mm sodium citrate and 100mm NaCl at pH 6.9


The following methods were applied during the study:

Visual inspection of particulate formation and quantification of turbidity changes by measuring changes in absorbance at 350nm before and after storage and/or agitation of samples in vials. Loss of protein due to aggregation was estimated by the decrease in absorbance of the solution at 280nm or by size-exclusion high-performance liquid chromatography (SE-HPLC) at 214nm and 280nm after centrifugation to remove insoluble material.

SE-HPLC was carried out on a Waters 2695 model liquid chromatography system using a GE Healthcare Life Sciences Superdex 200 column (1cm by 30cm). Protein elution was monitored at 214nm and 280nm, and the area under the protein peak was compared to controls that were stored in vials that had not been agitated. The elution buffer consisted of 20mm sodium phosphate and 150mm NaCl (pH 6.8).


The vials were stoppered and sealed with an aluminum crimp and placed horizontally on an orbital shaker at 200rpm at room temperature (RT) for up to 120 hours. The concentration of each protein was typically 1mg/ml unless otherwise noted. Vials were filled with 1.0ml of protein solution and all samples were run in triplicate.


For each protein, the absorbances at 350nm and 280nm were measured at the start of the experiment to determine protein concentration and to establish a baseline for turbidity measurements. After filling the vials, the remaining solution was stored in a glass screw cap vial at 4°C (‘stock’) until the experiment was completed. Controls consisted of storing filled vials at both 4°C and at RT without agitation. However, the absorbance of solutions stored at RT did not differ measurably from those stored at 4°C or from the stock.


Preliminary experiments indicated that mAb1 aggregated when shaken in glass vials. To develop a set of standardised test conditions, we determined the speed of the orbital shaker, which produced measurable aggregation and the optimal sample volume for aggregation.

Extent of Aggregation
When glass vials were mounted horizontally on the shaker, aggregation was negligible until the speed of rotation was increased to at least 200rpm. None of the molecules in this study aggregated below 200rpm. At this speed, the optimum sample volume was 1.0ml. These parameters are comparable to those used in similar studies (2). When the vials were mounted upright, aggregation did not occur.

These results are consistent with the notions that the dimensions of the liquid-air interface, as well as parameters such as the velocity of agitation, can affect the rate and extent of aggregation (2). By comparison with glass, aggregation of mAb1 in vials made of CZ under the same conditions was much less. Aggregation was also time-dependent (data not shown) and most of the studies were carried out for 96 hours.

Other Proteins Tested
Under the conditions established above, we examined the aggregation of two other therapeutic mAbs and rabbit IgG – a protein in the same structural class. Under the conditions of this study, rabbit IgG aggregates less in vials made of CZ than in those made of glass.

Following quantitation of the amount of soluble protein by absorbance at 280nm of the solutions after centrifugation to remove insoluble aggregates, it was clear that 94 per cent was soluble in CZ vials after shaking, while 60 per cent remained soluble in glass vials. Similarly, mAbs 2 and 3 aggregated to a lesser extent – or, in some cases, not at all – in vials made of CZ (data not shown).

Some proteins appear to be completely stable to aggregation when subjected to agitation under the conditions employed in this study. For example, a therapeutic fusion protein, which is genetically engineered to retain some structural features of a mAb, did not aggregate in vials made of glass or CZ (data not shown).

Surfactants are frequently added to biotherapeutic formulations in order to prevent surface denaturation (3). We tested whether Polysorbate 80 (PS80) (0.03-0.01 per cent, weight per volume) could prevent the aggregation of mAb1 when shaken in glass vials. At 0.03 per cent – the lowest concentration tested – PS80 blocked the aggregation of mAb1.

Although the addition of PS80 prevented aggregation in shaken vials, in related experiments the surfactant had no effect on dissociating preformed aggregates of mAb1 (data not shown). Thus, while the surfactant is able to compete with proteins at the vial surface and prevent nonspecific adsorption and adsorption-induced denaturation and subsequent aggregation, it cannot disrupt the hydrophobic interactions between the regions on protein molecules that are believed to be responsible for stabilising aggregates.


In general, proteins showed a reduced extent of aggregation in vials made of CZ compared to glass vials when subjected to vigorous agitation. Although the air-liquid interface plays a major role in mechanically induced aggregation, the surface properties of the primary container are also important to consider. Rotation at high speed on an orbital platform shaker is a simple model of mechanical stress to examine the effects of agitation on the aggregation of therapeutic proteins. In addition, this method can be used to evaluate different vials for storage and administration of biologics.

In a related study, we examined the stability of several proteins in prefilled syringes made of glass or plastic that had been subjected to gentle end-over-end rotation for several weeks. Similar to the findings reported here, proteins in prefilled syringes made of CZ also had reduced levels of aggregation, relative to those in prefilled syringes made of glass (4). Taken together, the two studies suggest that primary containers made of CZ offer a potential solution for the packaging of biotherapeutics that are found to be unstable in vials or syringes made of glass.

1. Rosenberg AS, Effects of protein aggregates: An immunologic perspective, AAPS Journal 8: ppE501-E507, 2006
2. Hawe A, Wiggenhorn M, Van De Weert M, Garbe JOH, Mahler H-C and Jiskoot W, Forced degradation of therapeutic proteins, J Pharm Sci 101(3): pp895-913, 2012
3. Wang W, Singh S, Zeng DL, King K and Nema S, Antibody structure, instability, and formulation, J Pharm Sci 96(1): pp1-26, 2007
4. Waxman L and Vilivalam V, Evaluation of end-over-end rotation/agitation of protein solutions in prefilled syringes made from glass or plastic as a preliminary indicator of protein aggregation, Protein Stability Conference, Breckenridge, Colorado, US, 2011

Daikyo Crystal Zenith® is a registered trademark of Daikyo Seiko, Ltd. FluroTec® is a registered trademark of West Pharmaceutical Services, Inc. Crystal Zenith® and FluroTec® technology  are licensed by Daikyo Seiko, Ltd.

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Lloyd Waxman joined West in 2008 as a Principal Scientist. Lloyd holds three patents and has contributed to more than 60 publications. His primary interests include protein biochemistry, development of techniques to analyse protein stability and the interaction of biologics with primary container systems. Lloyd received his BA and MA in Physics from Temple University. He earned his PhD in Biophysics from Harvard University and spent six years as a research scientist at Harvard Medical School. Prior to joining West, Lloyd spent 19 years in various aspects of drug discovery and protein analysis at Merck Research Laboratories.

Vinod Vilivalam is a Senior Director for Global Technical Marketing of Daikyo Crystal Zenith® and elastomers products at West. Vinod provides scientific and technical support, and leads various research alliances with academic and commercial institutions to characterise and develop solutions for unmet needs. He has also published various peer-reviewed papers. Vinod earned his MS and PhD in Pharmaceutics in 1993 and 1996, respectively, from Duquesne University, Pittsburgh, and successfully completed a two-year business management programme in 1999 at The Wharton School, University of Pennsylvania.

Dennis Liu is currently majoring in Biology at the University of Virginia, and expects to graduate in May 2014. He served as an intern in the Pharmaceutical Delivery Systems segment of West  Pharmaceutical Services, Inc in 2012, during which time the work on this article was performed.
Lloyd Waxman
Vinod Vilivalam
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