The New Dimension in Viral Inactivation

With conventional heating methods, heat inactivation of virus is an attractive non-invasive physical treatment for high value pharmaceutical proteins. A drawback is the limited resistance of proteins exposed to elevated temperatures for times up to several hours. In addition, efficient heat transfer is often difficult in larger volumes. In order to achieve an inactivation process that allows for appropriate validation temperature gradients must be overcome. Microwave technology provides HTST heat treatment of product fluids in a continuous flow at flow rates up to 80 l/h. This allows for the inactivation of virus while maintaining the integrity of the pharmaceutical product.

By using microwave energy and a specially designed heat exchanger to heat and cool fluids faster than any known method - in milliseconds rather than minutes - the UltraTherm® System achieves multi-log reduction of viruses and microorganisms in heat sensitive fluid materials such as biotechnically derived recombinant proteins, cell culture media, serum, plasma and plasma proteins. This multi-log reduction is accomplished without impairing the product's properties and without the limitations of irradiation, filtration or chemical methods. Delicate pharmaceuticals that cannot be autoclaved are particularly good candidates for UltraTherm treatment.

Microwave heating allows for a rapid heat transfer in a continuous flow. Sensitive products such as pharmaceutical proteins are exposed for 180-680 milliseconds to high temperatures, hold times at peak temperatures are reduced to 2-4 ms. Heat denaturation of the protein molecules does not occur even at temperatures far beyond the melting temperature determined by differential scanning calorimetry. Protein precipitation is the first denaturing effect which can be observed: an appropriate selection of a respective carrier fluid (buffer) is demanding and might contribute significantly to expand the operational temperature range. Large protein molecules like immunoglobulins can be treated at temperatures which are destructive for small non-enveloped viruses, e.g. SV-40. Smaller protein molecules like tPA are stable to temperatures which inactivate the Parvovirus, which is highly heat resistant.

The superiority of HTST microwave heating becomes most evident in comparison to conventional virus inactivation: even at "moderate" temperatures (<80^oC) which are applicable to heat sensitive proteins, a broad range of viruses including large non-enveloped viruses, e.g. Reo3, is fully inactivated (a range of viruses which is not accessible by solvent/detergent-, acid/pH3.0-3.9- or urea/3M- treatment).

UltraTherm® System Description

Microwave heating is performed using the 5 kW continuous flow UltraTherm.

The UltraTherm® System is an entirely self-contained, GMP compliant HTST microwave heating system with complete computer controls and safety interlocks. Pump, heat exchangers, valves, tubing and instrumentation are built on a mobile stainless steel frame with wheels. Stainless steel and disposable components ease maintenance.

Process Flow Diagram of the UltraTherm® System

The UltraTherm® System controlled by a Siemens PLC, is operated by the user through an advanced touch screen display featuring hierachical organized screens which provide all primary control parameters. System software protects against improper operation. The process screen gives a flow chart view of the system with most major parameters displayed in real-time.

The microwave energy is produced by a 5kW generator and led through a waveguide to the applicator. The applicator ("Cartridge") consists of a preheat section, a PTFE (polytetrafluorethylene) coil and a cooling section. The teflon coil is positioned inside the high intensity microwave field to achieve maximum energy transfer to the fluid. Residence time can be varied at a constant flow by application of coils with different path lengths (27", 50", 75", 100" coil). Hold times may be varied by flow rate. Flow rates of 35 to 80 L h^-1 are feasible. The final fluid temperature is monitored by a non-intrusive infrared temperature sensor and is controlled by adjusting the output power of the microwave generator.

Product is drawn through an autoclavable pump stage into the first heat exchanger where it is preheated. Subsequently it passes through the microwave field where the temperature is rapidly raised at 300^oC/sec, destroying viruses. Then the product flows into the second heat exchanger where it is rapidly cooled, halting product destruction. Products can be held at any target peak temperature from 60^oC to 160^oC for hold times as short as 2 - 4 milliseconds.

Precisely controlled valves allow switching between the product and a flushing solution to accurately control the recovery of the virus-inactivated product. The entire heat exchanger cartridge is disposable and easily replaced.

For investigational purposes as well as validation of virus inactivation an optional injection system is available. With the injection system trial runs can be arranged for several temperatures with as little as 500 ml of solution.

Performance of the UltraTherm® System

The UltraTherm was challenged under full scale continuous processing conditions for different peak temperatures (78^oC and 90^oC) over a period of 9-10 hours at a constant flow of 60 l/h.

The Siemens PLC provided full control and realized an extremely narrow range of processing temperature: 78+0.3^oC and 90+0.3^oC respectively.

Performance of the UltraTherm® System at 90^oC and 60 L h^-1

Performance of the UltraTherm® System at 78^oC and 60 L h^-1

Optimization of the HTST Heating Process

In order to achieve the most benefit from the HTST heat treatment, (maximum viral clearance while maintaining the product's integrity and quality, i.e. protein structure) there is a need to find optimal physico-chemical buffer conditions where the protein solution can be exposed to a maximum elevated temperature without increasing viral resistance.

Experimental work on the stabilization of protein molecules demonstrates the importance of a thorough screening for an optimal buffer / carrier fluid composition, including the evaluation of stabilizing additives.

A humanized MAb was applied to various compositions of phosphate and citrate buffers, differing in molarity, pH and conductivity. At a protein monomer level of 95 % the applicable peak temperature spreads over a range of 5^oC.

A humanized MAb was applied to a variety of buffers, differing in composition, pH, conductivity, some including an additive. At a protein monomer level of 99 - 100% the applicable peak temperature spreads over a range of 10^oC.

Influence of residence time on protein monomer preservationhuMAb2 at 60 L h^-1

Protein Structure
The tertiary structure defines the molecular shape of a protein molecule

• Tertiary structure arises from interactions between the side chains of the covalently linked amino acids forming the protein
• Tertiary structure orientates the critical residues and side chains into the correct geometrical relationship to permit function
• Hydrophobic interactions contribute greatly to the stability of the folded state

Denaturation of Proteins at High Temperatures
Loss of a protein's biological activity can occur by either conformational or covalent processes:

• covalent changes due to temperature, pH, chaotropic agents etc. may occur instead of, together with, or in addition to the unfolding phenomenon.
• the inactivation time course follows strict first order (unimolecular) kinetics, whereas aggregation phenomena are oligo- or polymolecular, and depend on protein concentration.

Stability of Protein Molecules at High Temperatures
Conformational stability of proteins is due to the (quite small) net difference between a very large number of weak stabilizing interactions and the nearly equally large conformational entropy.

• the net free (Gibb's) energy of stabilization is ca. 40 kJ mol^-1
• the stability of thermophilic proteins ( T 20-30^oC ) corresponds to a stabilizing G of 5-7 kcal mol^-1, and is simply due to a few additional interactions:
• 1 - 2 salt bridges
• several hydrogen bonds
• 7 - 10 CH₃ groups within the hydrophobic core

Stabilization of Proteins
Application of stabilizing additives

• low molecular weight substances are widely used to stabilize the protein's molecular shape and its biological activity
• the classification of additives falls into 3 general categories:
  • specific: substrates and specific ligands, where the native unfolded equilibrium shifts towards the native form
  • non specific: salts, polyhydroxy compounds, amino acids, fatty acids, phospholipids and surfactants
  • competitors: compete favorably with the protein for the inactivating agent, e.g. chelating and reducing agents

Stabilizing Additives

Salts
Hofmeister lyotropic series:

stabilising ions

(CH3)4N+>NH4+> K+, Na+>Mg2+>Ca2+>Ba 2+ SO42->Cl->Br->NO3->ClO3->SCN-

destabilising ions

Actions of stabilizing ions

• 'salt-out' hydrophobic residues causing the adoption of a more compact structure
• act via a surface tension effect
• act by shielding surface charges
• act as osmolytes by affecting the bulk properties of water
• salt ions contribute individually to exclusion and to binding, thus influencing protein hydration ( "Preferential Hydration of Proteins" )

Ammonium Sulfate

• contains two of the most stabilising ions: NH₄⁺ cation and SO₄^- anion
• stabilizes at 20 - 400 mM

other Salts

• Na₂SO₄
• citrate or acetate at 100 - 500 mM
• sulfate, phosphate or quaternary ions

Polyhydroxy Compounds ( Polyols )

• polyhydric alcohols ( e.g. glycerol, mannitol, sorbitol, xylitol ).The stabilizing effect is concentration dependent ( 10 - 40 % w/v ). Monohydric alcohols destabilize native conformation.
• carbohydrates (e.g. sucrose, maltose, trehalose) Maltose stabilizes plasma proteins ( globulins, plasminogen, antithrombin III ) against heat inactivation.
• Polyethyleneglycol (PEG) PEG may be used to final concentrations of 1 -15 % w/v

Amino Acids
have multiple effects in protein stabilization:

• Amino Acids (Gly, Ala) stabilize in the range of 20-500 mM
• may reduce the adsorption of the protein on surfaces (Gly)
• may prevent aggregation reactions (Glu, Asp at low pH; Lys and Lys/EDTA)
• may increase the melting temperature T_m ( e.g. Lysozyme T_m 68-80^oC; T_m/M: 4^oC/L-Pro, 7^oC/L-Ser, 6^oC/ß-Ala, 7^oC/Gly )
• stabilization effect of aliphatic amino monocarboxylic acids increases with carbon chain length ( e.g. plasmin ) but may induce electrostatic interactions with charged domains of the protein surface

Fatty Acids and Phospholipids

• thermal stabilization of serum albumin:
• aliphatic fatty acids of C₇ - C₈ had maximum affect
• DSC data: caprylate, N-acetyl-D,L-tryptophanate
• thermal stabilization of factor VIII:
• incubation with phospholipid results in 2-3 fold increase in coagulant activity

Surfactants
Proteins tend to concentrate at interfaces, causing precipitation due to irreversible unfolding.

• polysorbate ( Tween 20, Tween 80 ) at 0.02 - 0.5 %
• poloxamer 188 ( Pluronic 68 )

Chelating agents

• act to complex metal ions, hence prevent metal ion - induced aggregation
• prevent oxidation of thiol groups that is mediated by divalent metal ions: EDTA may be used to complex metal ions
• or may remove catalytically essential metal ion from the protein's active side, leading to loss of activity

Reducing agents

• prevent destructive oxidation of essential structural or functional features ( 0.5 - 1 mM DTT; 5 - 20 mM 2-ME )
• but can reduce disulfide bonds in proteins
• but can catalyze a thiol-disulfide exchange which may lead to aggregation
• CuCl₂ inhibits disulfide exchange: lysozyme inactivation at pH 6.0 / 100^oC not first order; with CuCl₂ significantly more stable, first order inactivation

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