May 1, 2014 (Vol. 34, No. 9)

R. Alexander Martino Fortebio
Mark Schofield Ph.D. Senior R&D Manager Pall Biotech
Rene Gantier Fortebio

BLI Provides Comparable Data at Higher Throughput,
Greater Speed, and Lower Cost

There are currently 35 monoclonal antibodies approved by the FDA as biotherapeutic agents, representing the most rapidly growing class of new drugs. Despite advances in downstream processing technology, affinity purification of monoclonal anti-bodies using Protein A chromatography is still the industry standard. In order to use Protein A resin as productively as possible it is important to load the resin at close to its dynamic binding capacity (DBC). The knowledge of DBC under these conditions allows us to scale our process robustly, while maintaining high resin productivity. 

To measure the DBC of Protein A for mAb, feedstock is loaded onto the column until binding sites for the mAb become saturated and mAb begins to break through. As contaminant proteins in the CHO feedstock have a large absorbance, it is impossible to accurately determine the breakthrough of mAb using UV A280. To follow the mAb breakthrough, we collected fractions of the column flow-through and analyzed the samples by Protein A HPLC and Protein A bio-layer interferometry assay, the latter using the Pall ForteBio Octet system. We compare these analytical methods using several metrics including process time, preparation time, cost per sample, dynamic range, precision, accuracy, limit of detection, and limit of quantitation. We show here that the Octet system provides a fast, accurate, and economical means of quantifying mAb, and hastens process development.

HPLC and Octet Methods

Samples for Octet analysis were diluted 1:10 in diluent buffer (1%/wt. BSA, 0.02%/vol. Tween 20 in PBS) to bring the sample concentrations below 300 µg/mL and probed using Protein A biosensors (Pall ForteBio, part no. 18-5010) on an Octet RED96 system (Pall ForteBio). Samples for HPLC analysis were 0.2 µm filtered by centrifugation (3,000g, 10 minutes) using Pall 0.2 µm AcroPrep™  96-well filter plates (Pall Life Sciences, part no. 8019). Protein A HPLC analysis was performed using a 100 µL Poros A 20 µm chromatography column (2.1 x 30 mm, 0.1 mL) (Invitrogen, part no. 2-1001-00) on a Shimadzu HPLC system.

For each assay, 6 replicates of 8 point standard curves were generated. For the HPLC assay, standards were prepared in loading buffer. For the Octet assay, standards were prepared in a 90% ForteBio Sample Diluent, 10% mock CHO feedstock solution. These standards were measured using different biosensors and at different stages in regeneration of these biosensors to simulate how unknown samples are measured on a 96-well plate.

The following statistical parameters were applied as described. The limit of detection (LoD) and limit of quantitation (LoQ) were determined according to the method from ICH 1996 “Validation of Analytical Procedures: Methodology”.

Application DoE and Procedure for Generating Samples

A two-factor, two-level factorial design was used to test for the effect of residence time and mAb titer on sorbent DBC. The experimental space is described in Table 1.

Purified mAb was spiked into depleted CHO feedstock at 1 and 2 mg/mL. The spiked CHO feedstock was applied to a 1 mL Protein A column (mAb Select SuRe, GE Healthcare) with 3- and 5-minute residence times. Flow-through from the column was collected in 1 mL fractions.

Results

Theoretical LoD and LoQ values for the HPLC assay were measured as 20.69 µg/mL and 29.58 µg/mL, respectively.  The Octet assay is more sensitive with an LoD of 0.11 µg/mL and LoQ of 1.32 µg/mL. For the purposes of our analysis, an arbitrary value of 10% total error was chosen as the cut-off point for either the upper or lower range of the assay. Accordingly, the dynamic range for the HPLC assay was determined to be 62.5 µg/mL to 5,000 µg/mL, an 80-fold difference, which spans 1.9 orders of magnitude. The linear dynamic range of the BLI assay was determined to be 1.3 µg/mL to 150 µg/mL, a slightly larger 115-fold difference, spanning 2.1 orders of magnitude.

In generating the data for this application, 220 samples and 48 standards were analyzed, along with 2 blanks for each Octet sample plate and 4 blanks total for the HPLC Protein A run. Not including programming time, and using the slowest process as a reference, the Octet assay overall was almost 9 times faster than the HPLC assay (212 minutes for Octet, 1,707 minutes for HPLC). The total cost including buffer, consumable and operator cost amounted to $2.87 per sample for HPLC with vials, which reduced to $1.21 per sample when using HPLC with microplates. In comparison, the Octet assay cost per sample was $1.15 with purchased sample diluent, and only $0.93 when preparing our own diluent, representing a cost reduction of >20%. This does not take into account column cleaning, regeneration or theoretical plate testing, which add to the overall cost per sample when using HPLC.


Table 1. Factors and Levels Used to Define Experimental Space

Determination of 5%, 10%, and 20% Breakthroughs

The two assays were used to analyze the fractions collected from the breakthrough curve experiment. The breakthrough curves (Figure 1) span a mAb concentration range from 0 mg/mL to 2 mg/mL. The 5%, 10% and 20% breakthrough values calculated using the Octet system very closely matched the values measured using HPLC. The DBC values measured by both techniques never differed by more than 2 mg mAb/mL sorbent. The highest capacities are measured at the high mAb titer and longest residence time, as expected.


Figure 1. Breakthrough curve obtained using Octet system. Red points represent 5%, 10%, and 20% breakthroughs as obtained from 1 mL fractions. Other breakthrough curves are not shown.

Conclusions

We have shown that the Pall ForteBio Octet RED96 system can be used to accurately quantitate mAb breakthrough values in the presence of contaminant host cell proteins. The greatest benefit in using the Octet system is decreased process time. Switching from HPLC to Octet RED96 system reduced the total time for analysis from >24 hours to <4 hours. Compared to HPLC analysis, the Octet system has a slightly larger dynamic range, and performs best at low concentrations with an almost 30-fold lower LoQ. This lower LoQ is an advantage for the Octet platform when considering that sample dilution can be used to effectively expand the upper range of either assay. Another advantage to the Octet system is the cost per assay, which offers greater than a 20% cost reduction in comparison to an equivalent HPLC assay.

ForteBio, A division of Pall Life Sciences

Mark Schofield
Senior R&D Engineer
[email protected]
www.fortebio.com

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