Establishment of the human albumin for electrophoresis Ph. Eur. BRP batches 3 and 4 181
Establishment of the human albumin for electrophoresis Ph. Eur. BRP batches 3 and 4 M.-E. Behr-Gross1, A. Daas1, A. Eulig-Wien2, S. Christians2 ABSTRACT Due to the diminished stocks of the 2nd batch of the European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for human albumin for electrophoresis, the European Directorate for the Quality of Medicines & HealthCare (EDQM) initiated in 2014 an international collaborative study for the establishment of two replacement batches. The study was run under the aegis of the Biological Standardisation Programme (BSP). Thirteen laboratories participated in the collaborative study to verify the suitability of the candidate reference preparations according to the Ph. Eur. monograph 0255 using the zone electrophoresis (ZE) method with either cellulose acetate and/or agarose as the testing medium. The candidate preparations were found suitable for the intended purpose and were subsequently adopted in June 2015 by the Ph. Eur. Commission as human albumin for electrophoresis BRP batches 3 and 4 with an assigned range for albumin of 93.8 % to 98.3 % of the total protein content.
KEYWORDS Albumin, Biological Standardisation Programme, Biological Reference Preparation, zone electrophoresis, collaborative study, European Pharmacopoeia.
1. INTRODUCTION The current human albumin for electrophoresis European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) batch 2 was established in 2008 during a collaborative study run by the EDQM, Council of Europe, under the aegis of the Ph. Eur. group of experts for human blood and blood products (group 6B). Due to dwindling stocks of this BRP, the Biological Standardisation Programme Steering Committee endorsed a project for the establishment of a replacement batch. The project leader for the study was Dr Stefan Christians from the PaulEhrlich-Institut (PEI), Germany. After a pilot study, it was decided to produce two candidate BRP batches using a commercial albumin preparation that was spiked with human normal immunoglobulin, distributed in aliquots of 1 mL and subsequently freeze-dried at the EDQM. A feasibility study was performed at PEI to pre-qualify the two newly prepared batches of candidate material. An international collaborative study was then undertaken to verify the suitability of the new BRP candidate batches to serve in the system suitability test for protein composition by zone electrophoresis (ZE) [1] in accordance with the specifications of the Ph. Eur. monograph for human albumin solution (0255) [2]. Participants were requested to test by ZE the purity of representative samples of the candidate and current BRP batches. A value for the albumin content corresponding to the measured range of content of the main albumin band, expressed
1 M.-E. Behr-Gross (corresponding author’s e-mail:
[email protected]), A. Daas, European Directorate for the Quality of Medicines & HealthCare, 7 allée Kastner, CS 30026, F-67081 Strasbourg, France. 2 S. Christians, A. Eulig-Wien, Paul-Ehrlich-Institut, Paul-Ehrlich-Straβe 51-59, 63225 Langen, Germany.
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in per cent of the total protein content obtained by ZE using strips of cellulose acetate and/or agarose gels, had to be assigned.
2. PARTICIPANTS Thirteen laboratories from 10 countries participated in the collaborative study including 7 public laboratories (Official Medicines Control Laboratories (OMCLs) and other public institutions) and 6 manufacturers. A list of participants is given in section 7. Each laboratory is referred to in this report by an arbitrarily assigned number not necessarily representing the order of the listing in section 7.
3. MATERIAL, METHODS AND STUDY DESIGN
3.1. Materials All of the collaborative study participants received 2 vials of the human albumin for electro phoresis BRP batch 2 (sample A) and 3 vials of each candidate BRP (cBRP3: sample B; cBRP4: sample C). The cBRPs were produced at the EDQM by mixing a solution of albumin at 20 % with a solution of immunoglobulin for intravenous (IV) administration at 10 % and with phosphate buffered saline. The resulting solution was aliquoted in glass vials and subsequently freeze-dried, rubber stoppered and sealed with aluminium caps. After production cBRP3 and cBRP4 were stored, at 5 °C ± 3 °C at the EDQM. Homogeneity of filling expressed as variation coefficient was 0.25 % for cBRP3 and 0.03 % for cBRP4 as tested by weighing a subset of vials before and after filling. The residual water content determined by coulometry is 0.3 % for both batches. Each vial when reconstituted with 1 mL purified water had a protein content of about 5 %. Prequalification of the batches at the PEI was performed by cellulose acetate and agarose ZE on freshly reconstituted samples (5 % solutions) and on the reconstituted solutions stored at +4 °C for 14 days.
3.2. Methods and design Participants used the analytical method described under ‘Tests: Protein composition’ in monograph 0255 [2]. General information on ZE provided in section 2.2.31 Electrophoresis [1] of the Ph. Eur. 8th Edition was followed for the study. Cellulose acetate and/or agarose were used as testing support. The working dilution used for the BRP and the cBRP was 2 % for cellulose acetate ZE. For agarose ZE, participants were requested to follow the instructions of the gel manufacturer. Participants were requested to perform 2 assays either by cellulose acetate electrophoresis or agarose electrophoresis or using both methods, on different days, each assay including 1 vial of the BRP2, 1 vial of the cBRP3 and 1 vial of cBRP4, carried out in triplicate (i.e. 3 lanes per sample). The assays for each sample were to be run concomitantly, if possible. If this was not the case participants were to keep the reconstituted solutions at +4 °C and use them within 14 days. (Based on the experience with batch 2 and on the prequalification of the candidate batches, the study protocol allowed storage of the reconstituted samples for up to 14 days.) In case of assay failure or invalidity, it was requested that participants perform a new series of experiments. Results were to be reported for each assay, expressed as the per cent of the total protein content of the main band obtained in the electropherogram (corresponding to albumin).
Establishment of the human albumin for electrophoresis Ph. Eur. BRP batches 3 and 4 183
4. RESULTS AND DISCUSSION
4.1. Reported and centrally calculated results Thirteen (13) laboratories reported results. Each laboratory carried out 2 independent assays using either the method by cellulose acetate or by agarose or both, with the exception of one laboratory which submitted 2 sets of results from 2 different operators per day. They are coded as 10A and 10B respectively. Three (3) laboratories carried out both the method by acetate cellulose and by agarose. Nine (9) laboratories carried out the method by agarose only and 1 laboratory carried out the method by acetate cellulose only. For each assay, the triplicate results per sample (3 lanes) were first combined by taking the arithmetic mean. Results from the two independent assays performed in each laboratory were then again combined by taking the arithmetic mean. The standard deviations (SD) were calculated for individual and combined assay results. Assays were assessed for validity by comparing the values obtained for the current BRP2 with the assigned range. Validity was also assessed visually to detect unusual patterns in the distribution. Overall means and standard deviations were calculated per method and for the pooled set of results. The proposed assigned range for cBRP3 and cBRP4 were established using standard statistics based on normal distribution theory. A complete overview of reported and calculated results expressed as the albumin content in percentage of total protein content of the main band is given in Table 1a and Table 2a (acetate cellulose ZE) and 1b and 2b (agarose ZE).
4.2. Validity of the assays In Table 2a and Table 2b the mean and SD of the triplicate lanes per assay are shown, as well as the mean and SD of the means per assay. The overall mean and SD of laboratory means is shown at the bottom of the tables. The two sets of results from laboratory 10 were first combined before combining them with the other laboratories. A graphical representation of the laboratory means per sample is provided as histograms in Figure 1. Each box represents the mean per laboratory (N=3×2). Numbers are the laboratory codes. Results obtained with the method by acetate cellulose are labelled with an asterisk whereas results obtained with the method by agarose are not labelled. Figure 2 shows two-way plots of the mean results for cBRP3 versus the mean results for cBRP4. The numbers are the laboratory codes and the diagonal line represents the line of identity. The closer the results are to the diagonal line, the more similar the two candidate batches are.
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BRP2
cBRP3
cBRP4
Boxes represent the mean results per laboratory. Numbers in the boxes are the laboratory codes.
Numbers are the laboratory codes. Each number represents the mean of the two triplicate assays (N=3×2).
Results obtained with the acetate cellulose method are labelled with an asterisk.
Results obtained with cellulose acetate are printed in bold and italic. Results obtained with agarose are printed in normal font.
Figure 1 – Histograms of mean results per laboratory (albumin content in %)
Figure 2 – Two-way plot cBRP3 vs cBRP4
Results for BRP2 vary from 96.3 % to 99.4 % with the method by acetate cellulose and from 94.2 % to 98.9 % with the method by agarose. Results for cBRP3 vary from 94.3 % to 98.9 % with the method by acetate cellulose and from 92.8 % to 98.5 % with the method by agarose. Results for cBRP4 vary from 94.9 % to 98.6 % with the method by acetate cellulose and from 93.7 % to 98.6 % with the method by agarose. These results show that there is a slightly wider spread of results for both candidate batches compared to the current batch, although in terms of reproducibility this difference is not significant (see below). As a measure of precision, the SD of the triplicate responses of the candidate batches is compared to the current batch. In 18 of the 34 assays the SD is greater for cBRP3 than for BRP2. In 19 of the 34 assays the SD is greater for cBRP4 than for BRP2. Neither is significant (P>0.05, two-sided binomial distribution) so it may be concluded that this study reveals no significant difference in precision between the use of the current and the proposed batches. As a measure of repeatability, the SD of the between-day variation of the candidate batches was compared with that of the current batch. In 11 of the 17 assays the SD is greater for cBRP3 than for BRP2. In 9 of the 17 assays the SD is greater for cBRP4 than for BRP2. Neither is significant (P>0.05) so it may be concluded that this study reveals no significant difference in repeatability between the use of the current batch and the proposed batches. As a measure of reproducibility, the overall SD of the between-lab variation of the candidate batches was compared with that of the current batch (F-test on ratio of squared SDs, excluding
Establishment of the human albumin for electrophoresis Ph. Eur. BRP batches 3 and 4 185
lab 6). No significant (P>0.05) difference between the candidate batches and the current batch is found.
4.3. Overall results per method The laboratory means per sample (both vials of BRP2/cBRP3/cBRP4 combined), are shown in Table 2a and Table 2b, grouped by method. The overall mean for the method by cellulose acetate is 98.0 % for BRP2, 96.3 % for BRP3 and 96.3 % for cBRP4. The overall mean for the method by agarose is 97.3 % for BRP2, 95.9 % for BRP3 and 95.9 % for cBRP4. The summary results are shown in Table 3.
4.4. Assignment of a range based on pooled results Overall it can be concluded that the difference between the candidate batches is negligible compared to the difference between laboratories and the difference between methods. Since both batches were produced from the same bulk materials it is therefore considered appropriate to pool the results and assign the same albumin content to both batches. This gives a mean albumin content of 96.06 % and a standard deviation of 1.11 %. With a coverage factor of 2 this gives a range of 93.8 % to 98.3 %. This represents a range of 4.5 % which is markedly wider than for the current batch which, with limits of 96.7 % to 99.2 %, has a range of 2.5 %. However, the current study would give a range from 95.8 % to 99.3 % for BRP2 which is a range of 3.5 % and therefore also somewhat wider than assigned.
4.5. Influence of storage after reconstitution and protein concentration The project leader provided data on storage time and temperature after reconstitution and on the protein concentration in the assay. The difference in results between start and endpoint were small compared to the overall standard deviation of all results. Thus storage for 14 days after reconstitution, as was the case for BRP batch 2, could be recommended without impact on the quality of the product.
5. CONCLUSIONS The study demonstrated that the two candidate BRP batches 3 and 4 were suitable for use for their intended purpose i.e. to serve in the system suitability test for protein composition by ZE [1] in accordance with the specifications of the Ph. Eur. monograph Human albumin solution (0255) [2]. Comparison between the 2 supporting media currently used for routine control and batch release purposes (cellulose acetate and agarose) showed an acceptable consistency with regard to the measured albumin content expressed as percent of total protein content. Given this outcome, the cBRP3 and cBRP4 were thus proposed for adoption by the Ph. Eur. Commission as human albumin for electrophoresis BRP batch 3 and batch 4 with the assigned range for albumin of 93.8 % to 98.3 % of the total protein content. The Ph. Eur. Commission adopted the candidate preparations in June 2015. Stocks of the adopted BRP batches (catalogue number H0900000) are stored at the EDQM at 5 °C ± 3 °C and shipped to users at room temperature with the recommendation that they be stored at 5 °C ± 3 °C upon receipt. The stability of these BRP batches will be monitored at regular intervals.
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6. ACKNOWLEDGEMENTS The authors wish to thank all participants for their valuable participation in this study. Simone Schlünder from Paul-Ehrlich-Institut, Germany, is gratefully acknowledged for performing the experiments for the feasibility study. The EDQM shipment and production unit staff, and notably Nathalie Mécréant and Sylvain Pierre, is gratefully acknowledged. Sally Woodward is acknowledged for expert secretarial and organisational assistance. This study was run in the framework of the Biological Standardisation Programme (project code BSP124). The Biological Standardisation Programme is financed jointly by the Council of Europe and the Commission of the European Union.
7. PARTICIPANTS (IN ALPHABETICAL ORDER BY COUNTRY) G. Elmecker-Plakolm, Octapharma, Austria L. Memic, Baxter, Belgium W. Stevens, Health Canada, Canada T. Vukman Kordic, Croatian Agency for Medicinal Products and Medical Devices, Croatia V. Lievre, Agence nationale de sécurité du médicament et des produits de santé, France A. Laulan, LFB Biomédicaments, France S. Christians, Paul Ehrlich Institut, Germany L.M. Meirinhos Soares, INFARMED, IP, Portugal C. Alonso Verduras, I. Rodrigo Castro, Agencia Española de Medicamentos y Productos Sanitarios, Spain N. Hosta, Instituto Grifols SA, Spain M. Edblad, Octapharma AB, Sweden K. Boegli-Stuber, Swissmedic, Switzerland S. Spycher, CSL Behring AG, Switzerland
8. ABBREVIATIONS BRP: Biological Reference Preparation; BSP: Biological Standardisation Programme; cBRP: candidate Biological Reference Preparation; EDQM: European Directorate for the Quality of Medicines & HealthCare; N/A: not applicable; OMCL: Official Medicines Control Laboratory; PEI: Paul Ehrlich Institut; Ph. Eur.: European Pharmacopoeia; SD: standard deviation; ZE: zone electrophoresis.
9. REFERENCES [1]
Electrophoresis, general chapter 2.2.31. Ph. Eur. 8th Edition. Strasbourg, France: Council of Europe; 2013.
[2]
Human albumin solution, monograph 0255. Ph. Eur. 8th Edition. Strasbourg, France: Council of Europe; 2013.
Establishment of the human albumin for electrophoresis Ph. Eur. BRP batches 3 and 4 187
Table 1a – Cellulose acetate electrophoresis: results (raw data) reported by the participating laboratories Albumin in % of total protein content BRP2 Lab 2 5 11 13
cBRP3
cBRP4
Day
Lane 1
Lane 2
Lane 3
Lane 1
Lane 2
Lane 3
Lane 1
Lane 2
Lane 3
1
98.0
98.3
97.5
96.1
95.9
96.9
96.6
96.1
96.2
2
96.3
96.4
97.6
94.3
96.0
95.6
95.7
95.5
95.4
1
98.9
98.0
98.6
98.4
98.2
98.1
98.4
98.2
98.2
2
98.6
99.0
99.0
98.8
98.9
97.9
98.1
98.0
98.6
1
97.0
96.9
96.9
95.1
95.1
95.5
95.8
95.4
96.0
2
97.0
97.1
96.9
95.3
94.8
94.8
95.3
95.0
95.4
1
99.0
99.4
99.1
96.3
96.5
94.6
95.8
94.9
95.2
2
98.7
98.8
98.8
95.9
96.3
96.0
96.0
95.6
95.0
Table 1b – Agarose gel electrophoresis: results (raw data) reported by the participating laboratories Albumin in % of total protein content BRP2 Lab 1 2 3 4 6 7 8 9 10A 10B 11 12 13
cBRP3
cBRP4
Day
Lane 1
Lane 2
Lane 3
Lane 1
Lane 2
Lane 3
Lane 1
Lane 2
1
98.2
98.8
98.6
95.9
95.4
95.6
96.0
95.9
Lane 3 96.0
2
98.3
98.4
98.5
95.6
95.3
95.8
95.8
95.7
96.2
1
95.9
97.0
96.9
95.7
95.4
95.5
94.7
94.2
95.4
2
95.6
95.2
95.6
92.8
93.8
92.8
94.1
93.7
94.6
1
98.9
98.9
98.8
98.4
97.9
98.5
97.5
98.6
97.7
2
98.6
98.6
98.5
98.0
98.4
98.4
97.7
97.9
97.6
1
97.0
96.7
96.9
95.0
95.9
95.0
94.8
95.3
95.0
2
97.1
96.9
96.8
94.9
95.4
95.0
95.3
95.3
95.5
1
94.9
95.8
96.2
95.4
95.2
94.3
96.0
94.4
94.1
2
96.7
96.3
94.2
95.0
94.7
94.4
94.8
94.2
94.1
1
98.5
98.3
98.4
97.6
97.4
97.1
96.7
97.9
98.0
2
98.2
98.4
98.4
97.7
97.4
97.4
96.9
97.9
98.0
1
97.0
97.7
98.2
97.2
96.9
97.1
97.3
96.9
96.6
2
97.7
97.3
97.9
96.7
96.4
96.3
96.0
96.6
96.9
1
96.8
97.2
97.1
96.0
95.7
95.4
95.9
96.1
95.9
2
97.1
97.0
97.2
95.7
95.5
95.5
95.5
95.7
94.7
1
97.4
97.3
97.1
96.8
96.7
96.5
96.5
96.4
96.2
2
97.3
97.1
96.7
96.9
96.7
96.5
97.6
96.2
96.2
1
97.5
96.9
97.4
95.7
95.7
96.2
96.1
96.1
95.7
2
97.2
96.9
96.9
96.6
96.7
96.5
95.9
96.1
96.4
1
97.1
96.8
96.8
94.3
94.5
94.6
95.2
95.2
95.1
2
96.9
96.8
97.0
94.5
94.6
94.4
95.4
95.2
95.2
1
96.2
96.9
96.9
95.1
93.4
95.8
95.6
95.6
95.3
2
97.0
96.5
96.9
95.9
95.8
95.7
95.9
95.6
94.9
1
96.4
98.8
98.3
96.1
95.6
95.9
94.7
95.8
94.6
2
98.3
98.3
97.1
95.5
96.0
96.4
95.3
95.2
96.3
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Table 2a – Cellulose acetate electrophoresis: summary statistics of results Albumin in % of total content BRP2 Per day Lab 2 5 11 13 Mean
Day
cBRP3 Per lab
Per day
Mean (SD)
Mean
cBRP4 Per lab
SD
Per day
Per lab
Mean (SD)
Mean
SD
Mean (SD)
Mean
SD
1
97.9
(0.40)
97.4
96.3
(0.53)
95.8
96.3
(0.26)
95.9
2
96.8
(0.72)
(0.82)
95.3
(0.89)
(0.71)
95.5
(0.15)
(0.54)
1
98.5
(0.47)
98.7
98.3
(0.14)
98.4
98.3
(0.14)
98.3
2
98.9
(0.21)
(0.28)
98.5
(0.53)
(0.20)
98.3
(0.29)
(0.01)
1
96.9
(0.06)
97.0
95.2
(0.23)
95.1
95.7
(0.31)
95.5
2
97.0
(0.10)
(0.05)
95.0
(0.29)
(0.19)
95.2
(0.21)
(0.35)
1
99.2
(0.21)
99.0
95.8
(1.04)
95.9
95.3
(0.46)
95.4
2
98.8
(0.06)
(0.28)
96.1
(0.21)
(0.19)
95.5
(0.50)
(0.16)
98.0
96.3
96.3
SD
(0.98)
(1.44)
(1.35)
n*
4
4
4
Establishment of the human albumin for electrophoresis Ph. Eur. BRP batches 3 and 4 189
Table 2b – Agarose gel electrophoresis: summary statistics of results Albumin in % of total content BRP2 Per day Lab 1 2 3 4 6 7 8 9 10a 10b 11 12 13 Mean
cBRP3 Per lab
Per day
cBRP4 Per lab
Mean (SD)
Mean
SD
Per day
Mean (SD)
Mean
Per lab
SD
Mean (SD)
Day
Mean
SD
1
98.5
(0.31)
98.5
95.6
(0.25)
95.6
96.0
(0.06)
95.9
2
98.4
(0.10)
(0.09)
95.6
(0.25)
(0.05)
95.9
(0.26)
(0.05)
1
96.6
(0.61)
96.0
95.5
(0.15)
94.3
94.8
(0.60)
94.5
2
95.5
(0.23)
(0.80)
93.1
(0.58)
(1.70)
94.1
(0.45)
(0.45)
1
98.9
(0.06)
98.7
98.3
(0.32)
98.3
97.9
(0.59)
97.8
2
98.6
(0.06)
(0.21)
98.3
(0.23)
(0.00)
97.7
(0.15)
(0.14)
1
96.9
(0.15)
96.9
95.3
(0.52)
95.2
95.0
(0.25)
95.2
2
96.9
(0.15)
(0.05)
95.1
(0.26)
(0.14)
95.4
(0.12)
(0.24)
1
95.6
(0.67)
95.7
95.0
(0.59)
94.8
94.8
(1.02)
94.6
2
95.7
(1.34)
(0.07)
94.7
(0.30)
(0.19)
94.4
(0.38)
(0.33)
1
98.4
(0.10)
98.4
97.4
(0.25)
97.4
97.5
(0.72)
97.6 (0.05)
2
98.3
(0.12)
(0.05)
97.5
(0.17)
(0.09)
97.6
(0.61)
1
97.6
(0.60)
97.6
97.1
(0.15)
96.8
96.9
(0.35)
96.7
2
97.6
(0.31)
(0.00)
96.5
(0.21)
(0.42)
96.5
(0.46)
(0.31)
1
97.0
(0.21)
97.1
95.7
(0.30)
95.6
96.0
(0.12)
95.6
2
97.1
(0.10)
(0.05)
95.6
(0.12)
(0.09)
95.3
(0.53)
(0.47)
1
97.3
(0.15)
97.2
96.7
(0.15)
96.7
96.4
(0.15)
96.5
2
97.0
(0.31)
(0.16)
96.7
(0.20)
(0.02)
96.7
(0.81)
(0.21)
1
97.3
(0.32)
97.1
95.9
(0.29)
96.2
96.0
(0.23)
96.1
2
97.0
(0.17)
(0.19)
96.6
(0.10)
(0.52)
96.1
(0.25)
(0.12)
1
96.9
(0.17)
96.9
94.5
(0.15)
94.5
95.2
(0.06)
95.2
2
96.9
(0.10)
(0.00)
94.5
(0.10)
(0.02)
95.3
(0.12)
(0.07)
1
96.7
(0.40)
96.7
94.8
(1.23)
95.3
95.5
(0.17)
95.5
2
96.8
(0.26)
(0.09)
95.8
(0.10)
(0.73)
95.5
(0.51)
(0.02)
1
97.8
(1.27)
97.9
95.9
(0.25)
95.9
95.0
(0.67)
95.3
2
97.9
(0.69)
(0.05)
96.0
(0.45)
(0.07)
95.6
(0.61)
(0.40)
97.3
95.9
95.9
SD
(0.95)
(1.19)
1.07
n*
13
13
13
Table 3 – Summary results and overall mean values (albumin content in %)
Cellulose acetate Agarose Overall mean value
BRP2
cBRP3
cBRP4
Mean
98.0
96.3
96.3
SD
0.98
1.44
1.35
Mean
97.3
95.9
95.9
SD
0.95
1.19
Mean
N/A
96.06
SD
N/A
1.11
Assigned Range (coverage factor 2) N/A = not applicable.
1.07
93.8–98.3
