Current and future technologies for the production of plasma protein-derived medicines

Introduction

The plasma-derived medicinal product (PDMP) is unique: multiple medicinal products, 15 of which are listed by the US Food and Drug Administration (FDA) (1), are manufactured from the same, human-derived, variable source material (2). As such, any process needs to provide the starting intermediates from which each individual product can be isolated. In contrast to pharmaceutical and most biopharmaceutical products, each product is not a single entity and is defined by the manufacturing process. Consequently, PDMPs are defined by a minimum purity of the target, impurity and safety profiles, in which basic pharmacopoeial requirements may include in-process reduction and/or maximum values for certain proteins. Such definition means that although products, such as immunoglobulin G (IgG), may have the same identifying label, they will not have exactly the same composition and will therefore be similar but different and not directly interchangeable (3). An added complexity is the production of certain products or concentrates, which by definition, have several active proteins, the ratio of which needs to be retained lot-to-lot. Coagulation factor products may also be stabilised by the addition of albumin. Furthermore, the human source factor entails a risk of virus transmission and the inactivation and reduction of both known, unknown and emerging pathogens in manufacturing is mandatory. Authorities such as the European Medicines Agency (EMA) and the FDA regulate the production and use of PDMPs, with the World Health Organization (WHO) maintaining global surveillance and recommendations. Significant guidance is provided by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH).

This review focuses on the current and potential future technologies for the separation and purification of plasma proteins that are currently in therapeutic use, and which are applicable to future plasma-derived products. Many potentially disruptive techniques have been developed in attempts to rectify the significant product losses that occur with ethanol precipitation. Many innovations have been adopted by the plasma fractionation industries and many discarded as they have not resulted in significantly improved yields, purity or safety. Viral reduction/elimination, developed in the 1990s, has had a dramatically positive impact on the safety of PDMPs and is not covered in this review. Similarly, clinical usage and the supply chain from plasma donation to patient is not addressed. A brief discussion of fractionation economics is included because of the changing pattern in the amount of plasma being used in the production of different products. Plasma fractionation is an industrial biomanufacturing enterprise requiring significant chemical engineering resources and the production scale now needs attention to sustainability, a subject outside the scope of this article as is digitalization and the use of artificial intelligence (AI).

Ethanol fractionation

In 1940, advisors to the US Armed Forces recognized the need for a blood plasma derivative to treat distant combat victims. Albumin, with its effect on osmotic pressure and therefore control of blood volume, became the target. EJ Cohn, who headed the Harvard Medical School Physical Chemistry Laboratory, and had researched amino acid and protein solubility for two decades was tasked with the development of an albumin product. Later in the same year Cohn was able to present a scalable, ethanol-based process for albumin and other variants to isolate further plasma proteins (4).

Before the introduction of purification starting in the early 1960s, methods based on differential interaction with solid phases, such as chromatography or physical fields, fractionation methods were based on differential solubility. Derived from theoretical studies and the definition of proteins as crystallizable (5), the Cohn laboratory determined that five variables, protein concentration, ethanol concentration, temperature, pH and ionic strength, were necessary to separate the plasma proteins into a sequence of useful fractions. Ethanol lowers the dielectric constant of the medium and causes ion-ion, ion-protein and protein-protein interactions in dilute solutions, keeping solubility constant over a wide range of conditions (6). In the sequence shown for Method 6 (7) (Figure 1), one of multiple schemes, the first ethanol precipitation step provides the starting Fraction I for fibrinogen, leaving IgG and albumin in supernatant I. Fraction II+III provides the intermediate for IgG. Fractions IV-1 and IV-4 were initially used to remove protein impurities before the final precipitation of albumin in Fraction V, but now provide the starting points for alpha₁-antitrypsin (AAT) and Antithrombin III. An IgG fractionation process from Fraction II + III with multiple precipitations was developed by Oncley et al. (8), and has been used for decades despite major product losses. Modifications using chromatographic purification have improved yields (see below). Although Factor VIII (FVIII) could be recovered from Fraction I, it is now purified from cryoprecipitate and prothrombin complex concentrate (PCC), Factor IX and C1 Esterase are also isolated prior to ethanol fractionation. Kistler and Nitschmann (K-N) at the Swiss Red Cross further developed Cohn’s Method 11, which was designed for large scale use, to reduce the ethanol consumption from ca. 2,000 litres of ethanol per litre of plasma to ca. 1,200 litres with a concomitant reduction of processing volumes by 2.2 to 1.7 times the starting plasma volume (9).

Figure 1 Generation of the main plasma protein intermediates, initially using coprecipitation followed by anion exchange adsorption steps for coagulation factors and C1 esterase prior to ethanol fractionation. IgG is derived from either Fraction II + III in the Cohn system or Precipitate A in the Kistler-Nitschmann scheme. AAT, alpha1-antitrypsin; IgG, immunoglobulin G; PCC, prothrombin complex concentrate.

Each of the precipitation steps in the Cohn/K-N processes was meticulously defined, see Table 1, with the addition of reagents and ethanol a critical step. Cohn used dialysis membranes for the slow addition and avoidance of locally high concentrations of ethanol and formation of precipitates that could be recovered by centrifugation and foretold the complex chemical engineering issues that awaited industrial production of PDMPs. Such issues have been discussed by Rothstein noting, for example, that precipitation is influenced by the processing mode (batch vs. flow), solid or solution reagents, reagent concentration, rate of addition and protein concentration as well as laminar or turbulent flow, tank geometry, agitator design, sparger design and placement, use of static mixers and control of temperature (10). Conditioning by holding and addition of flocculants and filter aids promote efficient separation of precipitates, as discussed in detail by Foster, a pioneer of continuous processing in plasma fractionation (11). Precipitation parameters profoundly influence both recovery and purity.

Ethanol fractionation is still the mainstay of the established industry, providing the starting points for the main products. Cohn’s original focus was to develop albumin but today the ever increasing need for IgG drives the industry which has invested significant effort in process intensification and optimization to improve recovery so that the industrial investment can be utilised.

Downstream processes

The term “downstream process” is well established in monoclonal antibody (MAb) and biological product processing to describe the purification and viral inactivation sequence following “upstream” fermentation or cell culture. In the production of plasma-derived products, the term similarly refers to the processing steps which contribute to purification and viral safety steps. In fractionation, upstream, thus, refers to the precipitation sequence leading to the intermediate products.

Purification steps

Downstream purification processes of Cohn intermediates rely heavily on different forms of chromatography and UF. Ion exchange resins are used to purify bulk proteins through capture or in a flow-through mode whilst removing trace protein impurities, separating aggregates from monomers and removing in-process reagents. Chromatographic steps may also be designed to reduce the potential viral load. Many examples of downstream processes have been reviewed by Johnston and Adcock (12). In contrast to low yield precipitation which demands product separation either by centrifugation or filtration (often with the use of cellulose or silica-based filter aids), classical, column-based, axial flow chromatography offers high product recovery, frequently well over 90% depending on the adsorption/elution mode (13). Matrices in use are frequently agarose or cellulose based or are synthetic, including acrylic derivatives and have been developed for high flow rates and to withstand harsh cleaning and regeneration conditions. Derivatization with anion, cation, mixed-mode ligands, and specific affinity ligands determines the efficiency and attainable purity over a single step. UF, using membranes of defined pore size, was introduced into fractionation as a method to remove ethanol from albumin but usage became ubiquitous for product concentration, salt removal and buffer exchange, also termed diafiltration (DF). These steps are frequently used in sequence as indicated in Table 2, noted as UF/DF. Concentration steps are used to reduce the processing volume and diafiltration to adapt the intermediate process solution to the following unit operation. UF/DF is used to modify the process solution to facilitate nanofiltration and in the final processing steps to achieve the desired formulation buffer.

Viral safety operations

Downstream processes include virus reduction/inactivation steps, either dedicated or as a benefit of partitioning in chromatography or precipitation. These operations are the main contributors to the safety of PDMPs, recently reviewed by Farrugia (23) in the pathogen safety tripod (24). The tripod describes the three complementary facets of PDMP safety from plasma donation to final product testing, where the manufacturing process is a major contributor to safety through dedicated reduction/elimination steps (25). Donor selection through nucleic acid testing (NAT) screening can contribute with a ~1 log₁₀ viral removal with a further reduction of ~3–5 log₁₀ per mL plasma with the elimination of donations in the viraemic phase. In processing with two viral inactivation steps the reduction factor ranges from >10 to >12 log₁₀, bringing the total clearance to >14 to >18 log₁₀. These steps contribute to the potential viral load in the final product container to less than 10⁻⁶ virus particles and the significant safety of PDMPs (26). The steps are summarized in the pathogen safety tripod.

As shown in Table 2 the most frequently used operations are solvent/detergent (S/D) treatment to inactivate lipid-enveloped viruses (27) and nanofiltration to remove non-lipid enveloped viruses, with pasteurization being used primarily for albumin. The original solvents and detergents used in the S/D method developed at the New York Blood Centre were tri-(N-butyl)-phosphate (TNBP) and polyoxyethylene-p-t-octylphenol (Triton X-100) (28). The latter detergent is linked to ecological concerns and the non-ionic, C11-15 secondary alcohol ethoxylate, Deviron 13-S9 detergent has been proposed as an alternative (29). Nanofiltration, introduced in the mid-1990s, is now a standard procedure using 15, 20 and 50 nm filters to remove non-lipid enveloped viruses and has a more than 20-year safety record (30). The roster of virus inactivation and removal technologies has been reviewed (31) with reference to the ICH Guideline Q5A(R1) from 1999 (32). It should also be noted that incubation with caprylic acid, frequently used in IgG manufacturing, is also an effective virucidal step. A low pH hold of final IgG has also, fortuitously, been shown to contribute to viral safety (33). Viral safety operations are well established in the large volume processing industry and have led to reliable safety of PDMPs. Viral reduction/elimination steps are included in the downstream processes.

Integrated processes

The integrated processes for many of the current intravenous and subcutaneous IgG products approved in the last decade have been reviewed by Buchacher & Iberer with an emphasis on process analytics (33), Buchacher & Curling on process variants (34), Buchacher and Kaur with a critical discussion of purity (35) and Bertolini (36) with the latest approval in 2023 (20) following what could be termed a “platform” process. The processes and process development described by these authors originate from the transition from intramuscular to intravenous products and further to the inclusion of hyaluronidase in subcutaneous products. In addition, the increases in concentration of final products to 10% and later to 20% has necessitated process changes. Each process underlines the difference between IgG products and their similarity as well as differences. The focus on IgG is driven by multiple indications and therefore demand and a consequent need for process and yield improvement. Process changes to simplify and add robustness/reproducibility in manufacturing at scale, increased purity without, for example the introduction of new thromboembolic effects whilst maintaining clinical efficacy and patient preferences require significant development: Lebing et al. reported a 70% reduction of processing time and a 50% increase in yield over a legacy IgG process (37). Wasserman et al. have shown how attention to detail in change of mixer speed on precipitate resuspension, “minor” pH changes and inactivation times can significantly affect yield in 13 steps of the IgG process. The changes increased bulk filterability and reduction of impurities, including fibrinogen, Factor IXa and Factor XII (38). In the downstream purification processes, the choice of chromatographic adsorbent is critical, exemplified in the improvements to the manufacturing process for FVIII/von Willebrand factor (VWF) (39).

There are many options for downstream process unit operations and although many different techniques have been investigated, chromatography and UF/DF remain and are likely to remain the mainstay of PDMP purification. These sequences enable pre- and post-viral inactivation steps.

Current challenges

Economic challenges

Whilst the demand for IgG continues to increase (40,41⁾, the demand for plasma-derived coagulation factors is decreasing and has done so since the introduction of recombinant alternatives in 1992. Even such alternatives are now challenged by the bispecific MAb emicizumab (42,43) now commanding ca. 25% of the haemophilia A market. The global uptake of this product, independent of the economic status (low- and middle-income countries to high-income countries) of the country (44) demonstrates the increasing use of a recombinant product over the plasma-derived alternative with revenues increasing by 41% in countries outside the USA, Europe and Japan (45). Consequently, plasma fractionator first litre revenues are falling, creating the need for innovation of new plasma-derived products; however, for such new moieties recombinant or other therapeutic options may be preferable. Relatively little coagulation product is now made from the first litre of plasma with every litre being processed for IgG. The continued production of albumin, now a commodity product with high consumption in the Chinese market (46) may not continue, leading to the burden of profitability on IgG revenues for the major fractionators unless compensated by new products. Thus, the economic challenge of plasma, raw materials and manufacturing cost of 50–57% (47) of the total enterprise cost remains and can only be reduced if the plasma cost continues to be high, by the reduction in processing costs and product yield of IgG.

First/last litre economics

The plasma industry situation, based on first/last litre economics (48), has been summarised by von Bonsdorff et al. (49), revealing the development and production conundrum for the fractionators as well as those contemplating new fractionation facilities: will plasma be used primarily for the bulk proteins albumin and IgG with a potential reduction of albumin revenue? To what extent will the haemophilia and other rare disease communities be served with recombinant or gene therapeutic alternatives? What new “first litre” products will emerge? Will new process designs target simply IgG production, in contrast to Cohn’s work where albumin was the target protein? Strengers also asked if IgG will need to carry the complete economic burden of fractionation with potential cost increases for patients (50). Will then minor, from a production point of view, proteins be made from dedicated plasma pools analogous to the manufacturing of hyperimmune IgGs or will side fractions be sufficient sources? Such questions imply, if for no other reason, the loss of IgG in current processes alone (9) that de novo technologies are to be sought.

Since the introduction of hybridoma and recombinant technologies, the plasma fractionation industry has faced challenges from competing product alternatives, particularly coagulation products, with the loss of product revenue. However, the use of coagulation factors continues to a small extent in developed economies. The economic driver in fractionation is IgG, particularly with revenues from high price markets. The need now drives the search for alternative, high recovery processes focused on IgG.

New and potential concepts for plasma product manufacturing

Process development of plasma protein isolation has recourse to an array of technologies from precipitation, crystallization, centrifugation, two-phase extraction, expanded bed adsorption (EBA), chromatography in all its many forms and filtration from bulk separation to sterile filtration. However, chromatographic methods dominate current processes with UF/DF unit operations contributing to optimal process flow but not directly to purification. Current development focuses on three areas—affinity chromatography with expanded bed separation, two-phase extraction and continuous electrophoresis being developed outside the core industry. Common to these efforts to improve fractionation processes is that they do not use ethanol. The Cohn and K-N sequences have survived 80 years because they have been integrated with purification/separation and viral clearance technologies that provide safe products across multiple jurisdictions. However, ethanol, despite being easily available and contributing to an aseptic fractionation environment has disadvantages in common with most precipitation techniques applied to complex systems such as plasma. The separation of the target protein from plasma is seldom complete with partitioning between supernatant and precipitate and the occlusion of protein in the precipitate. The separation of precipitates by centrifugation or by depth filtration with the addition of filter aids involves protein loss. The poor recovery of IgG in the ethanol sequence, coupled with the increasing demand and use of the product, has always driven process innovation and it is in the last decade that ethanol-free fractionation methods have been researched.

Different precipitants including sodium citrate, Rivanol^® (PEG) (51), solid phase electrolytes (52) and more recently polyacrylic acid (53) have been explored as alternatives to ethanol with the use of PEG surviving as a standard step in some IgG processes. Caprylic acid precipitation is used as an impurity removal operation with viral reduction properties. Major steps forward have been made with the inclusion of ion exchange operations for bulk protein capture or for impurity elimination. Reverse phase chromatography is used for the removal of process reagents such as S/D components. Affinity chromatography was introduced as a cascade of adsorption steps producing relatively high purity intermediates (54) or for the selective removal of low concentration protein impurities such as isoagglutinins (55), with hitherto restricted use for major plasma protein purification. However, immunoaffinity chromatography with immobilized monoclonal antibodies and heparin affinity separations have contributed significantly to the improved purity and activity of coagulation factors (see Table 2).

A comparison with MAb production

Downstream processes for MAb purification start with a major capture step using recombinant, immobilized Protein A. This single step, in a three-step purification process from cell culture supernatant, provides a protein with 90–95% purity and recovery >95%. In a conservative cost analysis, the capture step contributes ca. US$ 15/g purified protein (56). Protein A resins with dynamic binding capacities in the range 60–80 mg/mL are now available and if they could be used for polyclonal IgG capture would indicate a processing capacity of 10 litres of plasma per litre resin with a 1,000-litre column to process a 10,000-litre plasma pool in one cycle but at significant cost. However, Protein A, which binds to the Fc region, does not recognize IgG₃. Protein G, which binds to both Fab and Fc regions, has been investigated as a purification technology for IgG from Cohn fraction II + III. A detailed analysis of the performance of two commercially available affinity resins demonstrated purity issues. Economic calculations concluded that the technology in 2012 was inadequate for an affinity capture step to be considered viable (57). A potential alternative is the use of immobilized nucleic acid aptamers which has been demonstrated at laboratory scale for the purification of coagulation Factors VII, H, and IX at a purity of 98% (58). However, the technology is expensive and the application to IgG is distant. Peptide ligands from very large libraries of unique ligands offer a potential solution to finding a capture step dedicated to plasma IgG (41). Any affinity chromatographic step analogous to monoclonal capture needs to be viable not only from performance, targeting >95% purity and >95% recovery in a single step, but also have an acceptable cost contribution to the manufacturing process.

The potential of affinity chromatography

A less expensive mode of capture is presented in the form of multimodal (mixed mode) chromatography (MMC) which frequently combines ion exchange and hydrophobic interaction as well as thiol affinity and hydrogen bonding etc. and has been used predominantly for the polishing of MAbs, removing aggregates, fragments and host cell proteins but has wider implications in the purification of biotherapeutics (59). However, Protein A (still dominant) cost issues have driven the application of MMC to the harvest step in monoclonal antibody processes with concomitant reduction in the number of purification steps demonstrated in a proof-of-concept study. Binding capacities in the range 24–53 g/L were reported (60). This research indicates the potential for MMC as a capture step for polyclonal IgG if the appropriate ligand can be designed. Fan et al. have demonstrated dynamic binding capacities using a 2-mercaptopyridine-3-carboxylic acid (MPCA) ligand for polyclonal IgG in the range of 77–115 mg/mL in a nonwoven membrane format also indicating the potential for MMC in plasma protein purification (61). New MMC ligands and resin formats developed from multi-modal ligand libraries should contribute to the general availability of adsorbents, which can be explored as alternative capture operations for plasma IgG without ethanol precipitation and with reduced purification steps needed in downstream processes (62).

Expanded bed adsorption

Separation processes based on differential interaction with solid phases can be performed in the classical column mode or by holding the adsorbent carrying the target protein ligand from the product stream by using weighted or magnetized beads. EBA utilizing 6% agarose beads weighted with tungsten carbide particles to a density of 2.9 g/L has been derivatized with a mixed mode ligand for the capture of IgG. A cationic ligand modified to include benzoic acid (Mabdirect MM) had an IgG binding capacity of ca. 15 g/L (63). Feedstock is applied in upward flow mode to expand the bed and after capture the bed is settled and the product eluted in classical chromatographic form. Miller et al. (64) have developed a sequence after cryoprecipitation and PCC adsorption for the direct capture of IgG on Mabdirect MM. Eluted IgG is then diafiltered, incubated with caprylic acid and subject to anion exchange chromatography and collected in the flow-through. After diafiltration and nanofiltration the product is formulated and held at low pH. An average product yield from source plasma has been reported at 67.05%±5.1%. Of all new methodologies, this process is the most advanced with a phase III clinical trial (65) evaluation of a 10% intravenous product demonstrating positive results. Nonetheless, fixed bed chromatography is simpler to engineer and operate. The proprietary nature of the technology and relatively minor improvement of yield may limit use to the patent holder.

EBA is one method of separating the stationary phase adsorbent from the mobile phase in an effort to reduce pre-treatment prior to chromatography and thus improve purification efficiency. After an academic renaissance two decades ago, there is renewed interest in magnetic separations (66). In this case, adsorbents are rendered magnetic using, for example magnetite (Fe₃O₄) and amino-silanized ferrites and derivatised with Protein A/Protein G for IgG capture (67) or other functional ligands to capture the target solute. After batch adsorption, the adsorbed protein-magnetic bead is separated from the feed stock in a magnetic field. Lack of an appropriate mixed mode adsorbent, cost issues and a lack of appropriate bioprocessing equipment have hitherto hindered the adoption of this technology.

Aqueous two-phase extraction

The last decade has seen a revival of aqueous two-phase extraction (ATPE) applied to plasma fractionation. Vargas et al. (68) have described a system with an upper PEG (4% w/w) phase and a 13% (w/w) lower potassium phosphate phase. IgG is collected in the PEG phase and albumin in the salt phase. IgG, which precipitates in the upper phase, can be purified by caprylic acid precipitation and anion exchange chromatography to 92% at 70% recovery. Albumin yield from the lower phase is reported as 91% at a purity of 90%. An analysis of the strengths and weaknesses of ATPEs (69) indicates an advantage in processing high protein concentrations and particle loads and the potential for affinity partitioning. However, lack of scale up experience, full scale engineering and the time required for phase separation are likely to limit the industrial applicability of the method which essentially targets the recovery of only IgG and albumin.

Tangential flow electrophoresis

Tangential flow electrophoresis in combination with membranes in a cassette format has been applied to the isolation of single plasma proteins (70). The proprietary technology which uses specifically manufactured acrylamide membranes, requires complex engineering with issues such as heat generation/dissipation and scale up demanding numbering up the fractionation modules rather than scale up. Power consumption calls into question the sustainability of this form of fractionation. It is currently unclear if the methodology is applicable to the isolation of more than a single protein and what the requirements are for downstream purification: viral clearance steps need to be included in the absence of such studies. After more than two decades of significant investment and a lack of peer-reviewed publications documenting product characteristics and safety, it has become clear that this technology will not become generally applicable to plasma fractionation.

Process alternatives compared

Of the processes described above it is clear that affinity chromatography provides the most specific purification step, as a robust ligand can be designed to target a single protein and the matrix to which it is attached can be designed for harsh operating conditions. Affinity products are generally available from well-established manufacturers, although cost considerations may restrict their use. The EBA technology described above also uses affinity chromatography but in a more complex engineering mode. The ligands employed provide capture steps, but downstream purification is still needed, bringing the technology into question as a disruptive fractionation alternative. Aqueous two-phase systems are an interesting alternative for the separation of IgG and albumin but currently lack the specificity for other proteins, may use large amounts of PEG and are slow processes. No reports of their use in biological processes have been found. Tangential flow electrophoresis development is limited to one organisation, is proprietary, limited in scale requiring multiple, complex engineering units. Each of the technologies requires the additional viral clearance steps that are common to all current processes.

An immunoglobulin-focused process

Current discussion on alternative processes for the recovery of multiple plasma proteins focuses on methods that are ethanol-free and should thus obviate the most significant current fractionation losses in the trunk fractionation for IgG. Foremost is the potential for immobilized nanobodies (camelid, single-domain antibodies) expressed in Saccharomyces cerevisiae (71). One such antibody, caplacizumab has been developed for the treatment of acquired thrombotic thrombocytopenic purpura (aTTP), indicating the safety profile of such antibodies when used in purification. An affinity product, CaptureSelect/IgSelect which binds all four sub-classes of IgG through the Fc CH3 region at high selectivity has been developed and used to develop a specific assay for IgG during fractionation (72), showing potential as an IgG capture product in full scale fractionation. Low binding capacity of this Fc antibody compared to Protein A and cost issues for a ligand produced by fermentation may hinder the application of the product. However, a body of intellectual property (73) suggests significant interest in the fractionation community. Use of a dedicated IgG capture step with the common recovery and purity values of affinity chromatography could mean the development of an ethanol-free fractionation scheme, an example of which is shown in Figure 2.

Figure 2 Proposed, sample flow chart for ethanol-free plasma fractionation starting with cryosupernatant plasma and with immunoglobulin G as the prime protein target. After cryoprecipitation, which facilitates chromatographic purification, other important proteins can be obtained from the “flow-through” by current, established downstream processes.

A future process that does not rely on precipitation is more adaptable to continuous processing. Affinity chromatographic steps deliver two fractions without the use of gradient elution in a cyclic load, elute, wash/clean, and regenerate sequence and can be configured using multiple columns. The commonest format for which is simulated moving bed chromatography (SMB), a technology which is in use in many industries including pharmaceuticals. SMB improves utilization of the resin capacity and offers a continuous processing mode (74). Although complex from an engineering standpoint such technology may enable a small footprint facility and potentially the alternative of continuous manufacturing of IgG from full scale plasma batches.

Many “new” technologies are based on academic research into purification enabled by continued studies on the surface interaction of proteins, the introduction of matrices for protein chromatography and hydrophilic membranes dating from the 1960s. Material sciences have enabled new separation products designed for industrial application which now present alternative, ethanol-free scenarios to produce multiple PDMPs.

Conclusions

This article focuses on the current technologies used in PDMP production in the established, large scale, commercial industry and the potential for new technologies that are either disruptive or can be used to improve product recovery from waste fractions in existing processes. It is clear that this industry has a significant investment in installed manufacturing capability and that consideration of disruptive technology such as affinity capture using nanobodies, peptides or multi-modal ligands for IgG poses a critical decision with essentially the development and registration of a new product(s). Each manufacturer has registered products in multiple jurisdictions and a wealth of data supporting the efficacy and safety of each product in clinical use. Regulatory agencies, therefore, pose a significant hurdle to major process changes which will result in new product introductions. Entirely new processes will require significant analytical and pre-clinical data and clinical trials may be required. In the past, new, potentially disruptive technologies have not been adopted by the industry but have been incorporated into the main processing sequence where they will not affect already manufactured products. Similarly, new technologies described above may be adopted, especially for IgG if they contribute significantly to yield improvement. However, for countries looking at improved self-sufficiency in PDMPs and the alternative of establishing a fractionation plant, new, ethanol-free, continuous (or not) manufacturing technology is an option. Such projects need to consider the lengthy pathway from laboratory process to industrial production, clinical trials and regulatory approval. This paper does not attempt to cover the challenges posed by the development of recombinant, hybridoma and gene technology-based product solutions already approved or in development signalling a changing future.

Acknowledgments

None.

Footnote

Provenance and Peer Review: This article was commissioned by the Guest Editor (Jan Hartmann) for the series “Source Plasma” published in Annals of Blood. The article has undergone external peer review.

Peer Review File: Available at https://aob.amegroups.com/article/view/10.21037/aob-25-31/prf

Funding: None.

Conflicts of Interest: The author has completed the ICMJE uniform disclosure form (available at https://aob.amegroups.com/article/view/10.21037/aob-25-31/coif). The series “Source Plasma” was commissioned by the editorial office without any funding or sponsorship. The author is self-employed with his own limited company John Curling Consulting AB, and receives no compensation from his company for the writing of the article. The author has no other conflicts of interest to declare.

Ethical Statement: The author is accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. US FDA. Fractionated plasma products. Last accessed 06.09.25. Available online: https://www.fda.gov/vaccines-blood-biologics/approved-blood-products/fractionated-plasma-products
2. WHO. Information Sheet. Ensuring the Quality and Safety of Plasma Derived Medicinal Products. Last accessed 11.09.25. Available online: https://cdn.who.int/media/docs/default-source/biologicals/blood-products/infosheet-plasma-derived.pdf?sfvrsn=9b6a9ca9_4&download=true
3. Siegel JE. Immune globulins: therapeutic, pharmaceutical, cost, and administration considerations. Pharmacy Practice News, Special Edition. 2011. Available online: https://www.pharmacypracticenews.com/Monographs-and-Whitepapers/Article/05-23/Immune-Globulins-Therapeutic-Pharmaceutical-Cost-and-Administration-Considerations/70243
4. Cohn EJ. The history of plasma fractionation. In: Andrus EC, Bronk DW, Carden GA, et al., editors. Advances in military medicine, volume I. Chapter XXVIII. Boston: Little. Brown and Co., 1948:364-443.
5. Cohn EJ, Hughes WL Jr, Weare JH. Preparation and properties of serum and plasma proteins. XIII. Crystallization of serum albumins from ethanol-water mixtures. J Am Chem Soc 1947;69:1753-61. [Crossref] [PubMed]
6. Kistler P, Friedli H. Ethanol precipitation. In: Curling JM, editor. Methods of Plasma Protein Fractionation. New York: Academic Press; 1980:3-15.
7. Cohn EJ, Strong LE. Preparation and properties of serum and plasma proteins; a system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids. J Am Chem Soc 1946;68:459-75. [Crossref] [PubMed]
8. Oncley JL, Melin M. The separation of the antibodies, isoagglutinins, prothrombin, plasminogen and beta1-lipoprotein into subfractions of human plasma. J Am Chem Soc 1949;71:541-50. [Crossref] [PubMed]
9. Kistler P, Nitschmann H. Large scale production of human plasma fractions. Eight years experience with the alcohol fractionation procedure of Nitschmann, Kistler and Lergier. Vox Sang 1962;7:414-24. [Crossref] [PubMed]
10. Rothstein F. Technological problems in large-scale plasma fractionation. In: Sandberg H, editor. Proceedings of the international workshop on technology for protein separation and improvement of blood plasma fractionation. US DHEW Publication No. (NIH) 78-1422. 1977;15-26.
11. Foster PR. Protein precipitation. Formation of the solid phase and conditioning by acoustic vibration. In: Sandberg H, editor. Proceedings of the international workshop on technology for protein separation and improvement of blood plasma fractionation. US DHEW Publication No. (NIH) 78-1422. 1977;54-65.
12. Johnston A, Adcock W. The use of chromatography to manufacture purer and safer plasma products. Biotechnol Genet Eng Rev 2000;17:37-70. [Crossref] [PubMed]
13. Alstine JM, Jagschies G, Łącki KM. Overview of Alternative Separation Methods in Relation to Process Challenges. In: Jagschies G, Lindskog E, Łącki K, et al., editors. Biopharmaceutical processing. Development, design and implementation of manufacturing processes. Elsevier; 2018:207-20.
14. Chtourou S, Porte P, Nogré M, et al. A solvent/detergent-treated and 15-nm filtered factor VIII: a new safety standard for plasma-derived coagulation factor concentrates. Vox Sang 2007;92:327-37. [Crossref] [PubMed]
15. Dolia S, Verma S, Pawar A, et al. Quality attributes of plasma derived human fibrinogen (Fibrogen-I®). Plasmatology 2021; [Crossref]
16. Josić D, Hoffer L, Buchacher A, et al. Manufacturing of a prothrombin complex concentrate aiming at low thrombogenicity. Thromb Res 2000;100:433-41. [Crossref] [PubMed]
17. Hoffer L, Schwinn H, Josić D. Production of highly purified clotting factor IX by a combination of different chromatographic methods. J Chromatogr A 1999;844:119-28. [Crossref] [PubMed]
18. Simon TL, Kalina U, Laske R, et al. Manufacturing of plasma-derived C1-inhibitor concentrate for treatment of patients with hereditary angioedema. Allergy Asthma Proc 2020;41:99-107. [Crossref] [PubMed]
19. Lebing W. Alpha1-proteinase inhibitor: the disease, the protein, and commercial production. In: Bertolini J, Goss N, Curling J, editors. Production of plasma proteins for therapeutic use. Hoboken, NJ: John Wiley & Sons; 2013:227-40.
20. Kang GB, Huber A, Lee J, et al. Cation exchange chromatography removes FXIa from a 10% intravenous immunoglobulin preparation. Front Cardiovasc Med 2023;10:1253177. [Crossref] [PubMed]
21. Matejtschuk P, Dash CH, Gascoigne EW. Production of human albumin solution: a continually developing colloid. Br J Anaesth 2000;85:887-95. [Crossref] [PubMed]
22. Price H, Genereux M, Sinclair C. Hyperimmune Immunoglobulin G. In: Bertolini J, Goss N, Curling J, editors. Production of plasma proteins for therapeutic use. Hoboken, NJ: John Wiley & Sons; 2013:207-16.
23. Farrugia A. The Evolution of the Safety of Plasma Products from Pathogen Transmission-A Continuing Narrative. Pathogens 2023;12:318. [Crossref] [PubMed]
24. Turecek PL, Hibbett D, Kreil TR. Plasma procurement and plasma product safety in light of the COVID-19 pandemic from the perspective of the plasma industry. Vox Sang 2022;117:780-8. [Crossref] [PubMed]
25. Kreil TR. Building blocks of the viral safety margins of industrial plasma products. Ann Blood 2018;3:14.
26. Dichtelmüller H. Ensuring virus safety of plasma products. In: Bertolini J, Goss N, Curling J, editors. Production of plasma proteins for therapeutic use. Hoboken, NJ: John Wiley & Sons; 2013:361-8.
27. Hellstern P, Solheim BG. The Use of Solvent/Detergent Treatment in Pathogen Reduction of Plasma. Transfus Med Hemother 2011;38:65-70. [Crossref] [PubMed]
28. Horowitz B, Prince AM, Horowitz MS, et al. Viral safety of solvent-detergent treated blood products. Dev Biol Stand 1993;81:147-61.
29. Banerjee K, Antonello A, Johnson S, et al. Demonstrating the Effectiveness of an Alternative to Triton X-100 for Detergent-Mediated Viral Inactivation in Biomanufacturing. Biotechnol Bioeng 2025;122:1087-95. [Crossref] [PubMed]
30. Roth NJ, Dichtelmüller HO, Fabbrizzi F, et al. Nanofiltration as a robust method contributing to viral safety of plasma-derived therapeutics:20 yearsʼ experience of the plasma protein manufacturers. Transfusion 2020;60:2661-74. [Crossref] [PubMed]
31. Inouye M, Burnouf T. The role of nanofiltration in the pathogen safety of biologicals: An update. Curr Nanoscience 2020;16:413-24.
32. ICH Harmonised Tripartite Guideline. Viral safety evaluation of biotechnology products derived from cell lines of human or animal origin. Q5A(R1). Last accessed 18.03.25. Available online: https://database.ich.org/sites/default/files/Q5A%28R1%29%20Guideline_0.pdf
33. Buchacher A, Iberer G. Purification of intravenous immunoglobulin G from human plasma--aspects of yield and virus safety. Biotechnol J 2006;1:148-63. [Crossref] [PubMed]
34. Buchacher A, Curling J. Current manufacturing of human plasma immunoglobulin G. In: Jagschies G, Lindskog E, Łącki K, et al., editors. Biopharmaceutical processing. Development, design and implementation of manufacturing processes. Elsevier; 2018:857-76.
35. Buchacher A, Kaur W. Intravenous immunoglobulin G from human plasma – purification concepts and important quality criteria. In: Bertolini J, Goss N, Curling J, editors. Production of plasma proteins for therapeutic use. Hoboken, NJ: John Wiley & Sons; 2013:185-206.
36. Bertolini J. The purification of plasma proteins for therapeutic use. In: Simon TL, McCullough J, Snyder EL, et al., editors. Rossi's Principles of Transfusion Medicine. Hoboken, NJ: John Wiley & Sons; 2016:303-20.
37. Lebing W, Remington KM, Schreiner C, et al. Properties of a new intravenous immunoglobulin (IGIV-C, 10%) produced by virus inactivation with caprylate and column chromatography. Vox Sang 2003;84:193-201. [Crossref] [PubMed]
38. Wasserman RL, Garcia D, Greener BN, et al. Manufacturing process optimization of ADMA Biologics' intravenous immunoglobulin products, BIVIGAM® and ASCENIV™. Immunotherapy 2019;11:1423-33. [Crossref] [PubMed]
39. Mori F, Nardini I, Rossi P, et al. Progress in large-scale purification of factor VIII/von Willebrand factor concentrates using ion-exchange chromatography. Vox Sang 2008;95:298-307. [Crossref] [PubMed]
40. Robert P. The global demand for PDMPs: current scenario and future trends. The supply of plasma-derived medicinal products in the future of Europe. 2024. Last accessed 25.03.25. Available online: Available online: https://www.centronazionalesangue.it/wp-content/uploads/2024/05/1a.Robert-Final2-1.pdf
41. Belmonte M, Albiero A, Callewaert F, et al. Understanding supply sustainability of plasma-derived medicinal products: Drivers and consequences of shortages. Vox Sang 2025;120:754-64. [Crossref] [PubMed]
42. Curling J, Farrugia A, von Bonsdorff L. The past, present and future of blood plasma fractionation. Biologicals 2025;91:101849. [Crossref] [PubMed]
43. Mahlangu J, Iorio A, Kenet G. Emicizumab state-of-the-art update. Haemophilia 2022;28:103-10. [Crossref] [PubMed]
44. Annual Global Survey. World Federation of Hemophilia Report on the Annual Global Survey 2023. Table 17, p 80. Last accessed 25.03.25. Available online: https://www1.wfh.org/publications/files/pdf-2525.pdf
45. Marketing Research Bureau. Title. International Blood/Plasma News 2025;42:92.
46. Berman K. Feeding China’s growing appetite for human albumin. Biosupply Trends Quarterly 2019. Last accessed 25.03.25. Available online: https://www.bstquarterly.com/assets/downloads/BSTQ/Articles/BSTQ_2019-07_AR_Feeding-Chinas-Growing-Appetite-for-Human-Albumin.pdf
47. Jervelund C, Haanperä T, Siersbaek N. The impact of plasma-derived therapies in Europe. The health and economic case for ensuring sustainable supply. International Plasma Protein Conference, Lisbon, 20-21 June 2023. Last accessed 11.09.25. Available online: https://copenhageneconomics.com/wp-content/uploads/2021/12/copenhagen-economics_the-impact-of-plasma-derived-therapies-in-europe_june-2021.pdf
48. Goss N, Curling J. The economics of plasma fractionation. In: Bertolini J, Goss N, Curling J, editors. Production of plasma proteins for therapeutic use. Hoboken, NJ: John Wiley & Sons; 2013:451-60.
49. von Bonsdorff L, Farrugia A, Candura F, et al. Securing commitment and control for the supply of plasma derivatives for public health systems. I: A short review of the global landscape. Vox Sang 2025;120:114-23. [Crossref] [PubMed]
50. Strengers PFW. Challenges for Plasma-Derived Medicinal Products. Transfus Med Hemother 2023;50:116-22. [Crossref] [PubMed]
51. Steinbuch M. Protein fractionation by ammonium sulphate, rivanol and caprylic acid precipitation. In: Curling JM, editor. Methods of Plasma Protein Fractionation. New York: Academic Press; 1980:32-56.
52. Johnson AJ, Macdonald VE, Semar M, et al. Preparation of the major plasma fractions by solid-phase electrolytes. In: Sandberg H, editor. Proceedings of the international workshop on technology for protein separation and improvement of blood plasma fractionation. US DHEW Publication No. (NIH) 78-1422. 1977:380-404.
53. McCann KB, Van Alstine J, Martinez J, et al. Polyacrylic acid based plasma fractionation for the production of albumin and IgG: Compatibility with existing commercial downstream processes. Biotechnol Bioeng 2020;117:1072-81. [Crossref] [PubMed]
54. Burton SJ, Baines B, Curling J, et al. Sequential protein isolation and purification schemes by affinity chromatography. International Patent WO 2006/0223831 A2. 2 March 2006.
55. Hoefferer L, Glauser I, Gaida A, et al. Isoagglutinin reduction by a dedicated immunoaffinity chromatography step in the manufacturing process of human immunoglobulin products. Transfusion 2015;55:S117-21. [Crossref] [PubMed]
56. Łącki KM, Riske FJ. Affinity Chromatography: An Enabling Technology for Large-Scale Bioprocessing. Biotechnol J 2020;15:e1800397. [Crossref] [PubMed]
57. Hofbauer L, Bruckschwaiger L, Butterweck HA, et al. Affinity Chromatography for Purification of IgG from Human Plasma. In: Magdeldin S, editor, Affinity Chromatography. InTech, 2012.
58. Forier C, Boschetti E, Ouhammouch M, et al. DNA aptamer affinity ligands for highly selective purification of human plasma-related proteins from multiple sources. J Chromatogr A 2017;1489:39-50. [Crossref] [PubMed]
59. Halan V, Maity S, Bhambure R, et al. Multimodal Chromatography for Purification of Biotherapeutics - A Review. Curr Protein Pept Sci 2019;20:4-13. [Crossref] [PubMed]
60. Kaleas KA, Tripodi M, Revelli S, et al. Evaluation of a multimodal resin for selective capture of CHO-derived monoclonal antibodies directly from harvested cell culture fluid. J Chromatogr B Analyt Technol Biomed Life Sci 2014;969:256-63. [Crossref] [PubMed]
61. Fan J, Sripada SA, Pham DN, et al. Purification of a monoclonal antibody using a novel high-capacity multimodal cation exchange nonwoven membrane. Separation and Purification Technology 2023;317:123920.
62. Shekhawat LK, Markle T, Esfandiarfard K, et al. Next generation multimodal chromatography resins via an iterative mapping approach: Chemical diversity, high-throughput screening, and chromatographic modelling. J Chromatogr A 2023;1699:464018. [Crossref] [PubMed]
63. Gomes PF, Loureiro JM, Rodrigues AE. Adsorption equilibrium and kinetics of Immunoglobulin G on a mixed-mode adsorbent in batch and packed bed configuration. J Chromatogr A 2017;1524:143-52. [Crossref] [PubMed]
64. Miller D, Vanderlee G, Vaute O, et al. PlasmaCap EBA: An innovative method of isolating plasma proteins from human plasma. Vox Sang 2023;118:128-37. [Crossref] [PubMed]
65. Therapure Biopharma Inc. A Prospective, Open-Label, Multicenter Study of the Efficacy, Safety, Tolerability, and Pharmacokinetics of Therapure PlasmaCap IG in Adults and Children With Primary Immune Deficiency Diseases. clinicaltrials.gov; 2020 May Report No.: NCT03238079. Accessed 28.03.25. Available online: https://clinicaltrials.gov/study/NCT03238079
66. Franzreb M, Siemann-Herzberg M, Hobley TJ, et al. Protein purification using magnetic adsorbent particles. Appl Microbiol Biotechnol 2006;70:505-16. [Crossref] [PubMed]
67. Salimi K, Usta DD, Koçer İ, et al. Protein A and protein A/G coupled magnetic SiO(2) microspheres for affinity purification of immunoglobulin G. Int J Biol Macromol 2018;111:178-85. [Crossref] [PubMed]
68. Vargas M, Segura A, Herrera M, et al. Purification of IgG and albumin from human plasma by aqueous two phase system fractionation. Biotechnol Prog 2012;28:1005-11. [Crossref] [PubMed]
69. Soares RR, Azevedo AM, Van Alstine JM, et al. Partitioning in aqueous two-phase systems: Analysis of strengths, weaknesses, opportunities and threats. Biotechnol J 2015;10:1158-69. [Crossref] [PubMed]
70. Evtushenko M, Wang K, Stokes HW, et al. Blood protein purification and simultaneous removal of nonenveloped viruses using tangential-flow preparative electrophoresis. Electrophoresis 2005;26:28-34. [Crossref] [PubMed]
71. Arbabi-Ghahroudi M. Camelid Single-Domain Antibodies: Promises and Challenges as Lifesaving Treatments. Int J Mol Sci 2022;23:5009. [Crossref] [PubMed]
72. Mozgovicz M, Lingg N, Bresolin ITL, et al. Quantification of human intravenous immunoglobulin from plasma and in process samples by affinity chromatography. Prep Biochem Biotechnol 2025;55:217-22. [Crossref] [PubMed]
73. Method of purifying immunoglobulin G and uses thereof. Accessed 28.07.25. Available online: https://patentscope.wipo.int/search/en/result.jsf?_vid=P12-MDMX59-51234
74. Jagschies G. Continuous Capture of mAbs—Points to Consider and Case Studies. In: Jagschies G, Lindskog E, Łącki K, editors. Biopharmaceutical processing. Development, design and implementation of manufacturing processes. Elsevier 2018:527-56.