Monoclonal antibodies are central to therapeutic drug discovery and biomarker research, but their preparation for LC-MS analysis can present workflow challenges. Traditional purification methods can be labour-intensive, require multiple technologies and introduce variability that complicates downstream results.
Solid phase extraction (SPE) with positive pressure processing is becoming more prevalent in lab workflows due to its versatility and reliability for LC-MS sample preparation and can be leveraged for well-plate and column-based antibody preparation.
This poster presents AffinEx™ Protein A, an innovative solution that simplifies monoclonal antibody purification for easily scalable operation.
Download this poster to discover:
- How antibody purification can be automated using positive pressure processing
- How AffinEx Protein A can be cleaned and reused
- Why AffinEx is logistically advantageous
Qi Huang, Steven Alo, Heather Eastwood, Tuan-Linh Nguyen, Karsten Liegmann and John Laycock, Tecan SP, Baldwin Park, CA, USA
Scalable and automatable benchtop protein A based monoclonal antibody purification using AffinEx solid phase extraction columns with Resolvex® A200.
Introduction
Monoclonal antibody (mAb) research is one of the main forces behind the search for novel, targeted therapeutics for treating various diseases in recent years. Protein purification via affinity capture has proven a useful technology from benchtop purification to commercial manufacturing scales. Due to the diversity of sample types, protein contents and sample volumes, the technologies involved in benchtop sample purification are diverse, including gravity feed, vacuum processing, centrifugation, magnetic beads, Te-Chrom and FPLC. For different scales of purification, different technology is often used. The involvement of various technologies makes method transfer, training, scaling and automation challenging while requiring costly investments in capital equipment and the need for large lab space. In addition, agarose-based protein A sorbent, the most popular media, requires proper hydration during shipping and storage with refrigeration, which consequently results in added cost and complexity to both logistics and laboratory management.
Figure 1: Diverse forms of technology used for mAb purification.
Positive pressure sample processing (PPSP) provides a platform for a wide spectrum of workflows from manual operation to automation. The workflow is also readily scalable for protein loading and different sample volumes by working with solid phase extraction (SPE) cartridges in various sizes from 1ml to 6ml volumes.
The introduction of SPE columns filled with a rigid organic polymer-based protein A sorbent brings new opportunities to the classical protein science lab with an easily automatable workflow using Resolvex positive pressure processors. Owing to the ambient stability and mechanical rigidity of AffinEx, the sample preparation workflow and logistical considerations become simple, as depicted in this presentation.
In this presentation, we report IgG extraction and recovery rates using AffinEx Protein A columns in 4 different column formats, stability study results of AffinEx Protein A and potential reusability of these columns after repeated alkaline cleanings and renewals.
Figure 2b: SPE affinity extraction for mAb clean-up.
Figure 2c: Resolvex A200 integrated with a Freedom EVO® robotic platform.*
Column Format
NBE
1ml
3ml
6ml
Protein A volume (μl)
50
100
250
500
Loading volume (ml)
0.5
1
2.5
5
IgG loading
1
2
5
10
Recovery (%)
84
89
87
89
CV (%)
1.3
5.9
5.5
1.4
Table 1: Human serum IgG recovery from four column volumes.
Figure 2a: Resolvex positive pressure sample processors: M10, A100 and A200.
Four Protein A filled SPE column formats were tested. Columns were loaded with purified human serum IgG and are specified in Table 1. Purified human serum IgG in a PBS solution (2mg/ml) was loaded to each column format in a replicate of four. Following the extraction workflow, IgG recoveries from all four column formats were consistently between 84% - 89%. Sample loading was handled manually, while the rest of the workflow was done on a Resolvex A200-96 for NBE and 1ml formats and a Resolvex A100-48 for 3ml and 6ml. All four workflows were fully programmed. The extraction workflow is shown in Figure 3.
Human serum IgG recovery
Figure 3: AffinEx Protein A extraction workflow.
This test is designed to establish the shelf life of AffinEx Protein A SPE products under normal logistical conditions post-production (or production representative conditions) from the manufacturing site to the end user, following the Anderson and Scott protocol [1]. The goal is to develop a product with the stability to provide an adequate shelf life in an ambient lab environment (nominal at 23° C) while also considering the logistics involved in domestic and international shipping.
Stability study of AffinEx Protein A SPE columns
Figure 4: The Arrhenius Equation, the Q10 temperature coefficient is a measure of temperature based on the chemical reactions.
The last 4 entries of the recovery tests were performed on a Resolvex A200 integrated on a Tecan Freedom EVO liquid handling system*. All pipetting was handled via the Freedom EVO and the extraction workflow was carried out on the A200. The extraction results were identical to the results from the previous weeks demonstrating the robustness and scalability of the workflow.
AffinEx Protein A in NBE IgG (1mg) Recovery
Sorbent Lot 1
Sorbent Lot 2
Sorbent Lot 3
Entry
Week
Recovery %
St. Dev.
CV %
Recovery %
St. Dev.
CV %
Recovery %
St. Dev.
CV %
1
0
83.6
0.038
1.3
82.3
0.113
4
81.1
0.048
1.7
2
1
86.4
0.068
2.5
83.2
0.08
3.1
83.3
0.048
1.9
3
2
81.7
0.058
2.2
77.2
0.145
5.7
78.5
0.083
3.2
4
3
84.1
0.058
2.1
81.6
0.065
2.4
79.5
0.067
2.5
5
4
83
0.143
5.2
85.3
0.06
2
85.4
0.107
3.8
6
6
80.1
0.27
10.1
79.9
0.059
2.2
79.5
0.046
1.7
7
8
84.4
0.057
2.1
81.5
0.08
2.9
81.9
0.05
1.9
8
10
80.6
0.108
3.9
80.5
0.08
2.9
78.5
0.123
4.6
9
12
82.6
0.068
2.5
84.2
0.053
1.9
83.1
0.065
2.4
10
14
78.9
0.049
1.8
80.3
0.039
1.4
80.1
0.031
1.2
11
15
81.4
0.251
9.6
80.1
0.264
10.2
81.5
0.035
1.3
12
17
83.5
0.044
1.7
83.9
0.036
1.4
82.8
0.036
1.4
13
19
82.1
0.048
1.8
81
0.034
1.3
82
0.051
2
14
21
82.2
0.046
1.6
83
0.054
2
82.4
0.043
1.6
15
23
81.4
0.11
4
81
0.089
3.3
82.2
0.026
0.9
Mean
82.4
81.7
81.5
St. Dev.
3.75
4.1
3.87
% CV
4.6
5
4.8
Max.
86.4
85.3
85.4
Min.
78.9
77.2
78.5
Overall
Mean
81.8
Max.
86.4
St. Dev.
1.95
Min.
77.2
CV
2.4
Table 2: Performance of IgG recovery of NBE AffinEx Protein A after 23 weeks of storage in 37° C oven.
Conclusion.
AffinEx Protein A SPE columns provide a scalable workflow solution for mAb purification. With the Resolvex A200, the workflow may be automated to provide walk-away solutions for protein purification. Additionally, AffinEx Protein A columns are stable under ambient conditions for at least 24 months without refrigeration providing an economical solution for protein research.
Alkaline stability and reusability.
AffinEx Protein A SPE columns are stable for operation between pH 3-12. The columns may be reused after alkaline cleaning by flushing 0.1-0.5N NaOH solution through the sorbent bed, Columns may be regenerated after re-equilibrating the sorbent bed with a PBS buffer under a neutral pH. AffinEx Protein A in 1ml columns were tested for their functional performance after 20+ cycles of clean-in-place washing and renewal. No signs of deterioration in IgG recovery were observed.
AffinEx Pro. A 100μl 1ml column IgG recovery after alkaline washing and renewal cycles
Wash Cycle
0
7
23
IgG Loading
2mg
2mg
2mg
Recovery %
100.7
98.5
102.5
CV %
0.9
0.9
1.7
Table 3: Performance of IgG recovery
Figure 5: AffinEx Protein A alkaline washing and renewal protocol.
References.
1)
Determination of Product Shelf Life and Activation Energy for Five Drugs of Abuse, Clin. Chem. 37/3, 398-402 (1991)
*For research use only. Not for use in diagnostic procedures.
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