Advanced Liquid Particle Counters to Measure Aggregated Proteins

  Therapeutic proteins provide major health benefits (1), but aggregated proteins can have deleterious effects to both patient health and product efficacy. Protein aggregates are typically defined as self-associated species and are often classified by characteristics such as conformation, morphology, modification, reversibility, and particle size (2). Various techniques are now being used to quantify the size and concentration of aggregated proteins, including recently advanced liquid particle counters.

USP and FDA requirements and guidance
The new United States Pharmacopeia (USP) <787> Subvisible Particulate Matter in Therapeutic Protein Injections (3) test provides procedures to determine the quantity of particles present in a dose. The procedure is similar to USP <788> (4), with adjustments made to accommodate smaller product volumes and test aliquots. The particle size range (10 and 25 µm) for the pass/fail criteria remain the same as USP <788>. If the container volume is less than 100 mL, the limits are less than 6000 particles/container equal to or greater than 10 µm and 600 particles/container equal to or greater than 25 µm. If the container volume is greater than 100 mL, the limits are less than 25 particles/mL equal to or greater than 10 µm and 3 particles/mL equal to or greater than 25 µm. The smaller-volume products must also not exceed the per-container limits as defined for the greater than 100 mL injections. Dilution of the sample is allowed as long as the procedure is properly investigated and documented.

But measurements at 10 and 25 µm don’t necessarily characterize product differences of interest to the drug manufacturer trying to limit the degree of protein aggregation to the greatest extent possible. The FDA Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products (5) suggests that assessments should be made at 2–10 µm, and also in smaller size ranges (0.1–2 µm) as the methods become available. Note that no particle counting techniques are capable of detecting the monomer state of proteins that are typically in the 3–100 nm range. Dynamic light scattering (DLS) remains an analytical tool of choice in this size range.

Advances in particle counting

Light obscuration/extinction sensors have been used for 

USP

 <788> and <789> for decades. The original sensors typically operated across a dynamic range of approximately 2–400 µm or 1.5–150 µm with upper concentration limits approximately 10,000-15,000 particles/mL. The performance improved in the mid-1980s when the original white light source was replaced with laser diodes. The next advancement came when both the light extinction and scattering signals were incorporated into the calibration curve and reported results. The combination extinction and scattering sensors can now measure from 0.5–400 µm at concentrations up to 9000 particles/mL. The drive to measure at smaller particle sizes inspired the development of focused-beam sensors that improved small particle sensitivity and upper concentration limits. A focused-beam extinction sensor can now measure 0.65–20 µm at concentration limits 200 times higher than previously possible. Recently, focused-beam sensors detecting both extinction and scattering signals have broadened the possible dynamic range to 0.15–20 µm, also at 200x concentration limits.

The ideal particle counter for protein aggregation studies should be able to measure down in the 0.1-2 µm range suggested by the FDA guidance document and up to the 10–25 µm ranges required in 

USP

 <788>. The sample transport system should be able to perform analyses in sample volumes down near 100 µL and ideally conserve the sample for other measurements. Various new technologies have been introduced to fulfill this requirement including dynamic image analysis (microflow imaging) and resonant mass measurement (Archimedes). Because no single sensor technique is able to measure the entire range of 0.15–25 µm, a two-sensor approach has been introduced by Particle Sizing Systems (PSS), Port Richey, FL for protein aggregation studies (

Figure 1

). The first (lower) sensor incorporates focused beam extinction and scattering technology while the upper sensor uses a uniform beam extinction and scattering design. While the combination of these two sensors has a theoretical range of 0.15–400 µm, a practical upper limit of 150 µm exists due to the size of the liquid flow path through the smaller sensor. Hopefully, no therapeutic injections have particles above 100 µm, and scientists studying protein aggregation are far more interested in the 0.15–2 µm range.

Uniform beam sensors inspect 100% of the sample passing through the measurement zone. Focused beam sensors are capable of measuring smaller particle sizes because of the greatly increased light intensity created in the center of the liquid-flow path. The reduced inspection zone is also the reason for the higher concentration limits. The reduced zone decreases the chance of more than one particle at a time being present and interacting with the incident light. Because less than 100% of the sample stream is inspected, an algorithm is required to de-convolute the measured pulse heights into an accurate size distribution and concentration calculation.

Material and methods

Materials

Immunoglobulin G (IgG) lyophilized powder (Athens Research) was prepared at 1% weight percent by dissolving in PBS buffer and then diluted 100 to 1 to minimize coincidence effects during analysis. The PSS Nicomp 380 dynamic light scattering (DLS) system (Particle Sizing Systems) was used to determine the size of the nonaggregated IgG monomer (~15 nm) and aggregates (ranging from 200–600 nm and above). DLS does not quantify the concentration, only mean sizes.

System volume accuracy test

The system used for this study was first tested for count rate and volume accuracy by measuring a 15-µm count standard at a variety of sample volumes. The measurement procedure used is described as follows:
• An air gap of 0.05 mL was used in each run prior to sampling
• Flushes of 0.5 mL were performed before and after sampling (air gap took place after the ‘before’ flush, but before the sampling)
• A Tare volume of 0.15 mL
• Fresh 900 µL aliquots were used for each sample, regardless of sampling required.

Measurements were performed at the following sample volumes: 650, 550, 450, 350, 250, 150, and 50 µL. All measurements were performed in triplicate to also test reproducibility. The results from this study are shown in

Table I

and

Figure 2

.

Measurement procedure

A unique measuring process is necessary to meet all of the requirements for sizing and counting of aggregated proteins as described earlier in this article. In addition to the particle size range and small sample volume conditions, an additional challenge comes from the need to count enough particles for the result to be statistically valid while analyzing only 150 µL of sample. After many test samples were investigated, the best solution found is to pass the sample back and forth through the sensor multiple times to count at least several thousand particles. The uniform beam sensor inspects all particles at all times, but there are typically few particles >20 µm in a well-formulated sample. The focused beam sensor has a fraction of the inspection zone; therefore, although there are more small particles, multiple passes are required to build up an accurate distribution. A syringe sampler aspirates the sample from the original container up through the two sensors and then into a coiled tube above the second sensor. Once the sample container is empty, the flow is reversed, pushing the sample from the coiled tube back through the two sensors. This procedure is repeated several times until the desired particle count is achieved-typically in the tens of thousands.

Results

Before and after filtration

The purpose of these experiments was to track changes in the concentration in particles/mL of aggregated protein. The unfiltered IgG is shown in

Figure 3

in red. The x-axis is particle size in µm and the y-axis is number of particles/mL. The sample was then passed through a .45-µm syringe filter and analyzed again; represented by the blue result in

Figure 2

. The particle concentration >0.2 µm reduced from 5.43 x 10

⁸

particles/mL to 1.42 x 10

⁸

particles/mL by the filtration step.

Before and after heat stress

Another IgG sample was prepared and then diluted 250:1 prior to analysis. The sample was analyzed at room temperature and then heated by Peltier at 35 ºC for three hours to promote accelerated aggregation. The before and after heat stress results are shown in

Figure 4

. The concentration >0.2 µm increased from 9.75 x 10

⁷

particles/mL to 5.92 x 10

⁸

particles/mL due to the heat-stress process.

Conclusion

The measurement of aggregated proteins is becoming a more common practice in pharmaceutical laboratories. Several techniques are probably required to measure both the monomer and the aggregated size and to get accurate concentration data that are not available from light scattering techniques like DLS. Recent advances in liquid particle counter technology have improved the sensitivity to the range of 150 nm and reduced sample volume requirements down to ~100 µL, making this now a viable technique for both size and concentration measurements of aggregated proteins. This improved sensor design and small volume sample fluidics allow for measurements per

USP

<788> at 10 and 25 µm, as well as into the submicron region where more interesting aggregation phenomenon can be characterized.  

References

1. S. Aggarwal,

Nature Biotechnol.

28 (11), pp. 1165–71 (2014). 2. D.S. Dimitrov,

Methods Mol. Biol.

899, pp.1–26 (2012). 3.

USP

General Chapter <787>, “Subvisible Particulate Matter in Therapeutic Protein Injections” (US Pharmacopeial Convention, Rockville, MD, 2014). 4.

USP

General Chapter <788>, “Particulate Matter in Injections” (US Pharmacopeial Convention, Rockville, MD, 2012). 5. FDA,

Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products

(Rockville, MD, August 2014).   

About the Author

Mark Bumiller is regional sales and marketing manager, Particle Sizing Systems, Port Richey, FL,

mbumiller@pssnicomp.com

.   

Article Details

BioPharm International

Vol. 28, No. 4 Pages: 42–45 Citation: When referring to this article, please cite it as M. Bumiller, “Advanced Liquid Particle Counters to Measure Aggregated Proteins,”

BioPharm International

28

(4) 2015.