Upsala J Med Sci 99: 237-250, 1994 

5. Traceability of Concentration Values 

5.1 Establishment of a New Reference Preparation 
for 14 Plasma Proteins/CRM 470 = RPPHS Lot 5 

S. Baudner 

Behringwerke AG, Research Laboratories, 35001 Marburg/Lahn, FRG 

On January 1, 1990 the committee on Plasma Protein Standardization (PPS) of the 
International Federation of Clinical Chemistry (IFCC) was established. The members 
of this Committee are: 

J.T. Whicher, Leeds/Great Britain (Chairman) 
S. Baudner, Marburg/Germany 
J. Bienvenu, Lyon/France 
S. Blirup-Jensen, GIostrup/Denmark 
R. Ritchie, Scarborough/USA 

Further scientists working as associated members &d take part in this committee. 

Their task was to develop a new reference preparation for human plasma proteins 
which could be accepted worldwide and which would substitute the established WHO 
International Reference Preparations which had proved themselves as unsuitable for 
some modern immunoassays. 

5.1.1.1 Quality Requirements to the New Reference Material 

After some prelirmnary discussions it became the definite object of the Committee on 
PPS to reconcile the European and American tendencies as a basis recommendation 
for the standardization of plasma protein determination. Such a reference preparation 
would have t o  fulfil the following quality aims and claims: 

* all serum samples for the pool from healthy blood donors only with complete 
anamnesis and with established demographic constitution 

* absence of infectious diseases (certified) 
* serum pool immunochemically suitable for as many plasma proteins as possible 

237 



* physico-chemical characteristics of proteins clearly documented 
suitable for the majority of the commercially available immunoassays 
- method dependent bias very low - 

* longterm stability/long shelflife ( >  5 years) 
* suitable as an official calibrator 

* available in large quantities 

5.1.1.2 Milestones 

The above mentioned requirements and the studies to obtain the necessary stability 
data were accepted and initiated by the Bureau Communitaire de Reference of the 
European Communities (BCR, Brussels/Belgium) and by the College of American 
Pathologists (CAP, Northfield/USA), two institutions which have supported the IFCC 
project by financing various steps. 

Blood collection and the necessary characterization of the samples were organized to 
start in February 1990. The new matrix reference material was produced as a 
stabilized serum pool in December 1990. The filling and the freeze drying operations 
were performed in June 1991 after a previous successful trial using a pilot batch l o t  
0100/36. The final product was labeled with lot 91/0619. 

The freeze dried ampoules of the final product were divided into two groups with the 
following labeling: 

a. 

b. 

For the European Users (BCR) 
Commission of the European Communities 
BCR Reference Material for Proteins in Human Serum 
CRM NO 470 - CAP/IFCC Lot 91/0619 
for in vitro diagnostic use only/for 1 ml 

For the American Users (CAP) 
College of American Pathologists 
Reference Preparation for Proteins in Human Serum 
CAF'/BCR/IFCC Lot 91/0619 
for in vitro diagnostic use/for 1 ml 

A value assignment was performed for the following 14 plasma proteins: 

IgG, IgA, IgM, Albumin (ALB), Transferrin (TF) 
all mentioned in the German Quality Control Guidelines (RiliBAK) 

238 



Transthyretin (Prealbumin/TTR), al-acid Glycoprotein (AIAG), Haptoglobin (HPT), 
al-Proteinase-Inhibitor (q-Antitrypsin/AlAT), a2-Macroglobulin (MM), Ceruloplas- 
min (CER), C3, C4, CRP (by spiking). 

A detailed report of the IFCC Committee on PPS on the protein serum standard was 
released by BCR the material was named CRM 470, which stands for Certified 
Reference Material for immunochemical determinations of 14 human serum proteins. 

5.1.2 IFCC Material Lot 91/0619 - Blood Collection 

Human serum obtained from spontaneously clotted blood of healthy blood donors was 
used to prepare t h e  reference preparation for human plasma proteins. The human 
serum samples were provided by five different European blood centres: two actions for 
blood donation each from Sheffield and Danderyd (Table 1). All together there were 
seven different blood collections, carried out by: 

The Karolinska Institute, Danderyds Hospital, Danderyd/Sweden 
The National Blood Transfusion Service, Sheffield/Great Britain 
The Centre National de Transfusion Sanguine, Orsay/France 
Der Schweizerische Blutspendendienst, Bern/Switzerland 
Die Behringwerke AG, Marburg/Germany 

Haemolytic and lipaemic sera were discarded after an optical control. The preser- 
vatives sohum azide (0.5 g/l) and the proteinase-inhibitor aprotinin (80,000 KIU/l) 
were added to t h e  selected samples. Thereafter they were frozen by means of liquid 
nitrogen and stored at - 70°C for further use. Small quantities of the samples of every 
blood donation were stored separately and used for various analytical tests in 
accordance with t h e  "Requirements for Biological Substances/Reference Reagents" of 
t h e  WHO. A medical report was available for each blood donor. Only serum donations 
which fulfill the characteristics and requirements for "healthy blood donors" were 
further processed. The exclusion of monoclonal immunoglobulin classes IgG, IgA, IgM 
was also ensured. Data of concentration for cholesterol, triclycerides, glucose and GOT 
were obtained: they were all normal. The phenotypes of al-Proteinase Inhibitor and 
Haptoglobin were checked. Blood group, age, sex and race of the blood donors were 
listed. Serum samples containing factors which could possibly have an influence on the 
results of an immunoassay like rheuma factors ( >  30 IU/l), haemolytic components 
(Haemoglobin >40 mg/l) were discarded. Finally 364 blood donations (donors 20 to 60 
years old; 65% male) were found to be suitable and were used for the IFCC reference 
material. 

239 



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93
 



5.1.3 IFCC Material Lot 91/0619 -Stabilization, Filling Operation 
and Lyophilization 

Cholesterol 

1.85 
1.96 
1.91 
2.02 
2.05 
2.06 
1.81 

5.1.3.1 Requirements for Stabilization 
As discussed in a series of Committee meetings it was agreed upon that the 
stabilization procedure had to fulfil especially the following requirements: 

Triglyceride A p o  A-I A p o  B 

0.95 1.50 1.15 
1.16 1.50 1.10 
0.93 1.43 1.02 
1.30 1.43 1.15 
1.09 1.43 1.23 
0.96 1.50 1.15 
0.80 1.50 0.95 

* possibly complete elimination of the lipoproteins/lipids 
VLDL, IDL, LDL, HDL and Cholesterol 

* no or only minimal blank value - 
no or only minimal matrix effects 

* no or only minimal denaturation of the protein structure 
* no or only minimal reduction of the protein structure for the declared proteins 

Cholesterol 

- 
- 
- 
- 
- 
- 
- 

Treatment by Aerosila was the option chosen for the stabilization of the reference 
material. Behringwerke AG had t h e  task of performing this treatment. 

Triglyceride 

0.0157 
0.0167 
0.0121 
0.0121 
0.0163 
0.0157 
0.0096 

Table 2 

CRM 470 2 RPPHS Lot 5 
LipiddApolipoproteins before and after Stabilization 

single Pool 
Lot No. 

1 MR 0100/12 
2 BE 0100/18 
3 SH 0100/39 
4 SH 0100/40 
5 DS 0100/41 
6 DS 0100/42 
7 OR 0100/38 

single Pool 
Lot NO. 

1 MR 0100/12 

3 SH 0100/39 
4 SH 0100/40 
5 DS 0100/41 
6 DS 0100/42 
7 OR 0100/38 

2 BE 0100/18 

- not detectable 

24 1 



5.1.3.2 Influence of Treatment on the Blankvalue 

Llingle Pool 
Lot No. 

1 MR 0100/12 
2 BE 0100/18 
3 SH 0100/39 
4 SH 0100/4Q 
5 DS 0100/41 
6 DS Q100/42 
7 OR 0100/38 

The frozen blood donations were sent to Marburg at the beginning of December 1990 
where they were thawed and then pooled in seven different serum pools according to 
the place and date of the blood donation. The initial volumes of those single pools vari- 
ed between 7.1 and 10.2 liters. As had been agreed upon the C3 component was trans- 
formed into the stable C3c fragment by treatment with Inulin, after which each single 
serum pool was stabilized by treatment with Aerosil@. 

Blank Values Yield after Stabilization 
initial / final Volume Protein Cone. 
(Volt) / (Volt) (1) [g/l) 

4.55 / 0.64 5.7 63.2 
5.80 1 0.24 6.0 62.2 
5.77 1 0.78 6.5 69.8 
6.32 / 0.79 7.3 65.3 
4.47 / 0.39 5.0 64.6 
9.35 1 0.56 7.3 66.5 
1.82 1 0.62 5.2 69.6 

The success of the stabilization procedure was shown by the data regarding the blank 
values and yields as well as by the content of lipoproteins before and after stabilization 
(Table 2). The expected results were obtained for all serum pools. 

The successful removal of the lipoproteins, which tend to produce turbidity and 
matrix effects, can be proven on the basis of the drastically reduced blank values 
(Table 3): reduction to approximately 10% of the original value in the untreated serum 
pools, measured under undiluted conditions using a Behring Laser Nephelometer / 
BLN measuring the signals in Volt. 

Table 3 

CRM 470 RPPHS Lot 5 / Blank Value and Yield 

5.1.3.3 Influence of Treatment on the Protein Concentration 

It was necessary to concentrate the volume of the single pools after treatment with 
the stabilizing AerosilB. A n  average loss of volume of approximately 32% for the 
material had to be taken into account in order to obtain the original plasma protein 
concentration in the final pools. No great hfference in concentration before and after 
stabilization was found for the following eight plasma proteins: 

242 



IgG, IgA, Albumin, Transferrin, a2-Macroglobulin, Transthyretin (Prealbumin), acid 
a 1 - Glycopro tein, Hapt oglobin. 

A small reduction (< 10%) was observed for 
al-Proteinase Inhibitor (a1-Antitrypsin) and C3c. 

A clear reduction (on average 30%) was observed for 
IgM, C4 and Ceruloplasmin. 

Such reductions could not be avoided because the mentioned proteins also have a 
certain affinity t o  the stabilizing material, although it is not as strong as that known 
for lipoproteins. 

5.1.3.4 Influence of Treatment on the Protein Structure 

The protein structure - representative for the immunochemical reactivity - was also 
investigated using physicochemical procedures. It had t o  be proven that there were no 
structure alterations and no proteolytic digestions during stabilization. This was 
especially well recognizable on the basis of the completely intact structure for the 
protein inhibitors a 1-Proteinase Inhibitor and a2-Macroglobulin. The presence of 
intact protein inhibitors is very important because they represent a natural protection 
against any proteases possibly present in such preparations. Possible alterations of the 
protein structure were also checked for other proteins (section 5.1.5.4). 

5.1.3.5 Preservation of the Final Pool 

After fulfilling the set quality requirements for each single serum pool, the seven 
stabilized preparations were mixed into a single final lot (approx. 41 liters) on 
December 20,1990. For preservation/proteolytic protection 

* sodium azide (to final concentration of 0.5 g/1 

* aprotinin = Antagosan of Behringwerke AG (to final concentration of 80,000 
HU/U 

were added to the stabilized lot and this was immediately followed by sterile filtration 
and storage at - 20 "C/- 30 "C on the same day. The internal denomination IFCC ISRP 
lot 01 00/34 was given to the final bulk product (without CRP). 

1 7 - 955059 243 



6.1.4 IFCC Material Lot 01 00/36 - Pilot Batch 

Additives 

Sodium h i d e  

Bemamidine Chloride 

An aliquote of 1.0 liter of the final bulk product obtained on December 20, 1990 and 
stored at - 2OOC/- 30°C, was carefully thawed and used to produce a pilot batch on 
March 4, 1991. The following substances were added to this small trial batch for 
further experiments: 

Final Concentration 

mm. 15.4 mMol/l 

m a .  1 , O  mMol/l (< 0.157g/l) 
t< 1 g/o 

* a fraction containing CRP, lot 22 49 0 
(received from Prof. Pepys/Leeds) 
to obtain a final concentration of ca. 40 mg/l CRP 

* sodium azide solution (200 g/l) 
to obtain a final concentration of ca. 1 g/1 

* benzamidine chloride 
to obtain a final concentration of 0.157 g/1 

* HEPES buf€er to adjust the pH value to 7.2 

HEPES Bufer 

The pilot batch was filtrated under sterile conditions and 14 days later, on receipt of 
t h e  sterility protocol, filled into vials with 1.0 ml which were freeze dried and sealed. 

t o p H  7.2 

The sterility of the reconstituted lyophilisate was tested and once more confirmed. 
Further quality requirements regarding homogeneity, dry weight, rest moisture and 
especially the blank value of the lyophilisate after reconstitution with 1 ml destilled 
water were fulfilled within an acceptable range. The extremely low blank value, which 
can be measured in the reconstituted lyophilisate, indicated that freeze drying had no 
influence on the optical clarity of the material. 

Table 4 

CRM 470 A RPPHS Lot 5 / Preservation / Stabilizah'on 

I I Aprotinin I max S O , a X ,  KlU*/l 

244 



The stability testing program of the pilot batch was performed according to the 
instructions for stability studies and value transfer given by BCR. No significant 
alterations in protein concentration (measured by INA and RID) and protein structure 
(physicochemical analysis) were observed during the period of six months. An 
acceptable short-term stability of reconstituted material - up to seven days from the 
time of reconstitution - was also given. Results are identical or similar to those of the 
final product; therefore details for the pilot batch are reported in connection with the 
description of the certified preparation = CRM 470 (identical to RPPHS lot 5). 

STERILITY 
(af tor 
filling) 

HOHOGEWEITY 
(filling 
operation) 

DRY WEIGHT OF 
LYOPHILISATE 

REST HOISTURE 

B L A M  VALUE 
(BLW 

Table 5 

y e s ,  BWIEAB regulation y e s ,  BW/EAB regulation 

- Europaisches &rzneimittel Buch* 
1.003 mg (n = 5 )  1.0024 mg (n = 5 0 )  

SD = 6.4 mg 
CV = 0.639 % 

73.4 mg (n = 30) 71.8 mg (n = 3 0 )  
SD = 2.02 mg SD = 2.4 mg 
CV = 2.75 % CV = 3.34 % 
0.59 % (n = 30) 0.57 % H20 (Fischer) 
SD = 0.14 % SD = 0.11 % 
CV = 23.7 k CV = 19.3 % 

(n = 3 0 )  

OlOOf36 BY : 0.40 Volt 0100137 SY : 0.54 Volt 
(n = 3 0 )  (n = 20) 
(n = 3 0 )  (n = 20) 
0100/36 SL : 0.29 Volt 0100/37 SL : 0.46 Volt 

Pilot Batch 0100/36 SY and CRM 470 : RPPHS Lot 5 / 
Final Characteristics 

PILOT BATCH FINAL PRODUCT I Lot NO. 0100/36 SY CRM 470 2 RPPHS Lot 5 

~ ~~~~ 

S = Serum f Y = lyophil. f L = liquid 

* European Pharmacopoea 

245 



5.1.5 IFCC Material Lot 91 06 19 - Final Product 

5.1.5.1 G e n e r a l  R e m a r k s  

The final bulk product lot 0100/34 was thawed, spiked with the selected CRP fraction, 
protected with proportionally increased amounts of the previously reported 
preservatives (Table 4) and filtrated under sterile conditions. Vials were filled with 1.0 
ml, freeze dried under conditions identical to those of the pilot batch and sealed. The 
date of the filling operation was used to identify the reference material (ISRP lot 
91/06 19, i. e. June 19, 1991). The data obtained for characteristics (Table 5) like 
homogeneity, dry weight and rest moisture of the lyophilisate as well as the blank 
values before and after lyophilization (reconstituted samples) corresponded very well 
with those of the pilot batch. 

5.1.5.2 Stability Testing by Q u a n t i t a t i o n  b e f o r e  Reconstitution 

The stability studies were performed under the same conditions as for the pilot batch 
lot 0100/36/SY (SY = Serum lyophilized). The studies were performed according to a 
program proposed by B C R  

Storage T e m p e r a t u r e s  
- 70 "C/- 20 "C/ + 2 "C to + 8 "C/ + 20 "C (room temperature)/ + 37 "C/ + 45 "C 

T i m e  Intervals 
months: 1/2/3/6/9/12/18/24 (planned on: 36/48/60) 
(for the pilot batch shorter intervals/days: 1 /3/7/14/2 1 /28/56/ 126/192) 

Immunochemical Methods 
INA = Immunonephelometric Assay (BNA = Behring Nephelonieter Analyzer) 
RID =Radial Immunodiffusion (Partigen Plates of Behringwerke AG) 
After a storage time of 24 months the results indicated vary good stability (Table 6 for 
INA results). Exception: CRP for samples stored at + 37 " C /  + 45 "C. 

5.1 5.3 Stability Testing b y  Q u a n t i t a t i o n  a f t e r  Reconstitution 

The stability testing of the reconstituted material also indicated an excellent and 
stable quality of all proteins in the liquid form independent of storage at + 2°C to + 8°C 
or at +20 "C. Determinations were made daily up to seven days (short period) and to 
28 days (longest period) using a new vial each day. Results were compared to those 
obtained using a vial stored at - 70 " C .  N o  significant changes were observed for any of 
the proteins. 

246 



Conclusion: 

CRM 470 should be used within seven days after reconstitution with 1 ml distilled wa- 
ter when stored at + 2  "C to + 8  "C after each use. The stability over this period of time 
is very good, if contamination is avoided. 

Table 6 

C R M 4 7 0  = RPPHS5 1 
Stability Testing I Method: INA (BNAI 

Storage Time: 2 Years 
Results in Percent of Material stored at -7OOC 

4 7 0 0 3 C . x l ~  n = 2  

Reference: 
N-Protein Standard Serum, Ch.-B. 067651 
N-Protein Standard Plasma PY, Ch.-B. 068034 
NA-Latex CRP Kit, Ch.-8. 2361 3 
(Behringwerke AG) 

247 



5.1.5.4 Stability Testing by Analysis of Protein Structure 

The stability studies regarding the protein structure were performed under the same 
conditions as those for the quantitation of proteins by RID and I N A  i. e. by using 
samples stored at different temperatures and by testing within the same time 
intervals. The patterns were compared with those performed immediately after 
manufacturing. The methods used were: 

* Vertical polyacrylamid gel electrophoresis (PAGE) 
(P = 8.0%) with and without sodium dodecyl sulfate (SDS) 
to study the structure of various serum proteins. 
No significant alteration was observed. 

4: Vertical PAGE (P = 5.5%) for analysis af A2M 
showed that it mainly existed in the intact (slow) form and no changes to the fast 
form were observed during the period of testing. 

* Vertical PAGE followed by immunoblotting for CRP: 
The stored samples kept their intact form (exception: + 37 "C/ + 45 "C). 

* Vertical SDS PAGE followed by immunoblotting 
for C3/C3c: The existence of the fragment C3c was confirmed. 
for C4/C4c: The complete form C4 as well as small amounts of fragment C4c were 
detected. 
for A1AT The inhibitor-active and intact form corresponded t o  the majority of the 
protein present. 

* Two-dimensional immunoelectrophoresis 
for C3/C3c: All patterns were similar; no further alteration was noted after the 
initial fragmentation (nearly 100%) to the stable form of C3c and C3d. 
for - C4: 
for AlAT: The majority was present in the intact form together with a small 
amount of aggregates. 
for Slight degradation of this protein was seen (this is usual after 
lyophilisation). 

Only one peak corresponding to the intact form of C4 was observed. 

Summary of Examination 

No distinct alterations in protein structure (Table 7) were found during the period of 
stability testing up to 24 months referring the samples stored at - 70 "C/- 20 "C/ + 2 "C 
to 8 "C/+18 "C to +22 "C. 

248 



Table 7 

PROTEIN 

CRM 470 RPPHS Lot 5 
for 14 Plasma Proteins 

IFCC/BCR/CAP Material Lot No. 91 1061 9 

STRUCTURE STATE 

P R O T E I N  S T R U C T U R E S  

190 

I9A 

Igx 

intact f native 

intact f native 

intact; + aggregates 

CRP 

ALB I intact 1 native 

intact 

TRP I intact 1 native 
c3c fragment ++--- c4 mented slightly frag- 

TTR (PRE) 1 intact 1 native 
&, AG I intact 1 native 
a PI 
d t A )  

+ aggregates 
inhibitor active 
+ types 
intact (8-form) 
inhibitor active 

slightly altered 
siae unchanged 

HPT 
HP 1-1 
HP 2-1 I- mixture 
HP 2-2 (n = 364) 

METHOD FOR DETECTION * 

IE; 2-dim.IE 

IE; 2-dh.IE 

IE; 2-dim.IE 

IE; 2-dh.IE; PAGE18 % PAA 

IE; 2-dh.IE; PAGE18 % PAA 
~ ~~ 

IE; 2-dim.IE; PAGEISDS + IB 
IE; 2-dim.IE; PAGEfSDS + IB 

IE; 2-dim.IE; PAGE18 % PAA 

~~ 

IE, 2 - d h .  IE; PAGEISDS 
7 % PAA +- IB 

IE; 2-dh.IE; PAGEf5#5 % PAA 

IE; 2-db.IE 

IE; 2-dim.IE 

IE; 2-dim.IE; PAGE18 % PAA + IB 

2-dim. IE = two-dimensional Immunoelectrophoresis 
PAGE = Vertical golyacrylamide Eelelectrophoresis 
PAA = - Polyqcrylqmide 
BDS = Sodium Dodecyl Sulfate 
IB = Immunoblotting 

Bd/Pla / b:tb93-016.da Sgterrkr 2, 1493 

249 



Exceptions / Slight Alterations 

CER for samples stored at +37 "C and +45 "C after 18/24 months 
CRP for samples stored at + 37 "C and + 45 "C after 18/24 months 

Such results of physicochemical analysis confirmed t h e  data of the quantitation by im- 
munoassays. 

250