Purification approach for blood products

Feb 24,2025

Bestchrom

According to Chinese Pharmacopoeia (2020 edition), blood Products are defined as therapeutic products derived from human blood or plasma, such as human albumin, human immunoglobulin, human coagulation factor, etc. As far as the current situation of blood products in China is concerned, the blood products usually refer to plasma protein products.

Protein accounts for 7-8% of human plasma (content is between 60-80g/L). Nowadays, it is known that human plasma contains about 200 types of plasma protein components with unique biological functions. In order to make efficient use of this precious resource, modern technology is available for the isolation and purification of more than 20 types of plasma protein components. Blood products are available via strict virus inactivation. The excellence in safety, effectiveness and accuracy during clinical prevention and treatment makes it impossible to be replaced by other drugs.

Due to the ever growing demand for blood products, the market has been under huge pressure for the expansion of purification capacity. Various  purification methods have been involved in the purification of blood products, among which cold ethanol fractionation is regarded as the basic method of large scale plasma purification. However, as the separation technology innovates, chromatographic technology has witnessed a growing significance in the purification of plasma, among which ion exchange chromatography resin and affinity resin are most widely used in the particular sector.

Commonly used purification approaches

1. Cold ethanol fractionation

When temperature is low, solubility of proteins can vary in ethanol of different concentrations. Different components can be obtained via change in concentration, temperature and pH value of ethanol. After centrifugating the precipitates, it is possible to repeat the above mentioned operation on the supernatant and precipitation.

2. Ion exchange chromatography(IEX)

Ion exchange chromatography functions via the different surface electric charges carried by different proteins. After performing desalting or cold ethanol fractionation on plasma, load treated plasma components and let it flow through the IEX resins, achieving desired purification purpose by binding and elution of proteins via solution condition alteration(eg.pH, salinity). IEX can be applied for most blood products as a result of its advantage in endotoxin removal.

Affinity chromatography(AC)In affinity chromatography, protein separation is mainly achieved via specific adsorption among bio-molecules. Target protein can be purified by resins coupled with specific ligands(eg. Heparin,lysine and gelatin etc). Thus, for plasma proteins containing Fc-tag or antibodies, it is common to purify with Protein A affinity resins, which achieving both high purity and high yield.

3. Size exclusion chromatography(SEC)

This method achieves purification of proteins based on different molecular weights of proteins. This method is usually used in the desalting(first step) and polishing(last step) , effectively removing HAV viruses.

4. Expanded bed adsorption(EBA)

Combining features of both fluidized bed chromatography and adsorption chromatography, run components in upflow at set flow rate in column, where small molecules flow through before adjusting conditions including flow rate, pH and salinity to sequentially elute components with different molecular weights and charges. This is how the “first layered, then separated” effect is achieved.

Generally speaking, the application of chromatography technology has fundamentally overcome the shortcomings of traditional blood products purification methods including cumbersome operation, time-consuming process, unsatisfying performance and low level of automation. Therefore, plasma usage efficiency and finished products quality will be dramatically improved, providing more blood products options for the market.

The following flowchart shows plasma protein purification process via cold ethanol fractionation and chromatography methods

Fig.1 Plasma protein purification process via cold ethanol fractionation and chromatography methods

Chromatography schemes for various plasma proteins

1. IV human immunoglobulin (4^th generation) chromatography

Immunoglobulin (IgG) is the most abundant protein in blood other than albumin. IgG accounts for 75% of total immune globulins in blood. IgG is the major participant in humoral immune response. As the major molecule involved in body’s anti-infection, it has defensive functions including bind to pathogens/toxin, activating complement, strengthening phagocytosis as well as allergic reaction activation and transplanted organ rejection alleviation functions. Therefore, IgG is the most widely used immunoglobulin  in clinical application.

Human IgG has molecular weight of 150KD and pI from 5.8-7.3. The preparation of IV human IgG has witnessed persistent optimization. Take IV human IgG(3rd generation) as an example, cold ethanol fractionation + AEX method will leave too much impurities such as IgA and agglutinin, leading to clinical adverse reactions.

By contrast, take the IV IgG (4th generation) developed by Chinese biopharmaceutical company for example, cold ethanol fractionation + two steps of AEX+ anti-A and B hemagglutinins chromatography method can effectively improve product purity while still lowering risk of allergic reaction and hemolysis via removal of albumin, IgA, IgM and anti-A and B hemagglutinins. During the process, Octanoic acid precipitation+low pH incubation +20nm nanofiltration method is used for removal/inactivation of viruses to ensure the viral safety. Specifically, Bestchrom Diamond Q resin is used for the purification of IV IgG (4th generation) and enjoy excellent performance under pressure/flow posed by viscous feed like plasma, which contributes to easy scale-up, high binding capacity(nearly 200mg/mL) and high yield higher than 95%.

Fig.2 IV IgG (4^th generation) Chromatography process

2. Prothrombin complex concentrate(PCC) and human coagulation factor Ⅸ chromatography

Prothrombin complex concentrate (PCC) is compound of several types of coagulation factors. PCC mainly contains four types of vitamin K-dependent coagulation factors, namely prothrombin (FⅡ), anti-hemophilia B factor (FⅨ), prothrombin (FⅩ) and prothrombin transforming factor (FⅦ). Therefore, it is widely used for acquired deficiency of coagulation factors (FⅡ, FⅦ, FⅨ and FⅩ) relevant diseases. At present, processing-scale PPC purification mainly adopts cold ethanol fractionation + AEX method to purify plasma cryoprecipitate reduced.

As a very important participant in endogenous coagulation process of human body, a reduction in human coagulation factor Ⅸ content and its activity will enable the normal coagulation in human body, leading to hemophilia B. Human coagulation factor Ⅸ has molecular weight of 56KD and pI higher than 5. Plasma cryoprecipitate reduced is usually used in its purification process, which first uses DEAE Bestarose A-50 resin to bind factor Ⅱ, Ⅶ, Ⅸ, Ⅹ.

After buffer changing, Diamond DEAE resin is used in B/F mode , specifically, low salinity buffer can wash away partial coagulation factors with weak binding, after which, crude factor Ⅸ can be eluted by increasing salinity of buffer. The crude factor Ⅸ then will go through steps including UF/dialysis buffer change, S/D treatment, viral inactivation  and  purification by Bestchrom Heparin affinity resins before getting the final products. The activation of finial product can reach a level higher than 110 IU/mg.

Fig.3 Prothrombin complex concentrate(PCC) and human coagulation factor IX chromatography process

3. Antithrombin Ⅲ (ATⅢ) chromatography

The antithrombin ATⅢ is the most important physiological anticoagulant protein in human body, accounting for about 70% of the whole active anticoagulant system, as well as participating in maintaining the dynamic balance of coagulation and anticoagulant system. Heparin sodium can bind with ATⅢ and enhance its inhibitory effect on activated coagulation factor Ⅱ, Ⅸ, X, Ⅺ and Ⅻ. Therefore, Heparin resins are commonly used in the purification of antithrombin ATⅢ by purifying Ⅰsupernatant or Ⅳ precipitation, getting high purity finished products via one-step process. 

Fig4. Antithrombin III (ATIII) chromatography process

4.C1 Esterase inhibitor chromatography

C1 esterase inhibitor can inhibit complement, fibrinolysis, coagulation and kinin while exerting 90% OF FVIIa inactivation function in human body. Currently, processing scale preparation method uses IEX+HIC resins for the purification of plasma cryoprecipitate reduced. Specifically, use plasma cryoprecipitate reduced as raw material. After using Bestchrom CM Bestarose FF resin for the initial purification, use Phenyl Bestarose FF(HS) resin for further purification. Desalt the purified sample, remove virus by Pasteurization and nano-membrane filtration. Finally get finished products after preparation, aspetic filtration and freeze-drying.

Fig5. C1 esterase inhibitor chromatography process

5. Human serum protein chromatography

Being the mostly abundant and important proteins in serum, human serum proteins account for 50%-60% of the serum composition and possesses important physiological function by maintaining nutrition and osmotic pressure. HAS is a single chain sugar-free glycosylated protein. It consists of 585 amino acids with molecular weight of 66.5kD and pI from 4.7-4.9. In 1946. Edwin J.Cohn developed the Cohn process(Cold ethanol fractionation) for the isolation of albumin. In 1962, Kistler and Nitschmann from the Swiss Red Cross made improvements on Cohn process and established Kistler-Nitschmann fractionation methods. After that, human albumin preparation technology develops continuously, where chromatography and filter pressing technologies improves impurity and yield of albumin. The virus inactivation technology further ensures the safety of human blood albumin.

Blue Bestarose FF is affinity resin for albumin purification. Based on agarose matrix, the resin uses Cibacron Blue 6GA as its ligands and enjoys advantages including excellent physical&chemical stability, low ligand leachability, long service life and wider application. In addition to specific binding, the resin can also bind target proteins via unspecific binding such as charge and HIC approaches.Nowadays, Blue Bestarose FF resin has been widely applied in the purification of various proteins including dehydrogenase, kinase, transferase, serum albumin, interferon and plasma protein, as well as the capture of plasma-sourced albumin or recombinant proteins via specific binding.

6. Other plasma protein chromatography

The preparation of human coagulation factor VIII: use cryopercipitation as raw material, filter by Tris buffer, absorb target molecules by Al(OH)₃ gel or powder, process by S/D, use AEX resin for purification, conduct processing on eluate to get finished product.

The preparation of human fibrinogen: use the eluted fractions from factor VIII preparation (AEX purification step) as raw material, ECH-Lysine Bestarose 4FF resin can be used for the purification of human fibrinogen.

7. Recombinant proteins

Recombinant plasma protein products are developing rapidly. Therefore, output of clinically important coagulation factors such as Ⅶ, Ⅷ, Ⅸ has exceeded that of blood-derived products. For recombinant plasma purification (e.g Fc fusion factor VIII), process used for antibody can serve as a reference for its purification. To be more specific, Protein A affinity + AEX+CEX/HIC approach. Bestchrom resin has been successfully used in Fc fusion factor Ⅷ clinical scale up cases. Ni affinity resin can be used for His tagged protein, whereas non-tagged recombinant protein can adopt IEX+HIC+MMC approach for purification.

Conclusion：

Considering the ever growing demand for blood products, it is urgent to improve capacity and efficiency of blood product production. Among various purification methods being used for blood blood production, cold ethanol fractionation serves as cornerstone for scale-up of plasma protein production. Nevertheless, it is still worth noting that chromatography technology is enjoying increasing importance, among which IEX and affinity chromatography resins are widely employed. Bestchrom provided multiple chromatography resins for the efficient purification of blood products, facilitating quality improvement and productivity boosting of relevant producers.

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