As a final nail in the coffin to the claims of purification/isolation of the Polio “virus,” for the “safety and effectiveness” of the Salk Polio vaccine, as well as the purification methods of Carlton Schwerdt (who it was later admitted only achieved purity of 10% of the “virus”), I present two studies from 1961, which place them 8 years after Salk’s vaccine research and 7 years after Schwerdt’s initial “purification” of the Polio “virus.” The studies both come from Jesse Charney, a man who actually worked with Schwerdt and supposedly developed further improvements on his purification methods. In fact, according to one source, Charney achieved the greatest purification of Polio.
From the book Coriell: The Coriell Institute for Medical Research and a Half Century of Science:
That one blurb from the above source is all I can find in the history books on Polio purification/isolation regarding Jesse Charney. Any further information on the man besides coming across some of his work is nonexistent. That seems rather odd for a man who achieved “the greatest purification of Polio.” Instead of Charney getting any accolades, the praise goes to Loring asnd Schwerdt for their 1% and 10% purification efforts. Maybe looking at some of Charney’s work will help to solve this mystery.
First up is a study from July 1961 which details a new purification method devised by Charney using nucleic acid precipitation. This method is supposed to further purify the “virus” from tissue cultures in order to eliminate any monkey kidney contaminates that may be present from the process. At the time, there were theories that the monkey kidney cells could cause nephrotoxicity (rapid degradation of kidney functions) when given to people in repeated injections with multiple vaccines made from monkey kidneys. It seems rather obvious now that animal tissues should not be injected directly into the bloodstream of humans but apparently it wasn’t so back then:
The Concentration and Purification of Poliomyelitis Virus by the Use of Nucleic Acid Precipitation
“The precipitation of poliovirus by nucleic acid at low pH represents a new, highly satisfactory method for the concentration of virus from large volumes of tissue culture filtrate. This method has been used as a first step in a procedure for the purification of poliovirus. Virtually pure preparations so obtained have permitted the redetermination of the extinction coefficient of poliovirus (E&i = 81.6) and the determination of the specific refractive increment of the virus (An = 0.00174). Sufficient quantities of the virus became available through the use of this procedure for the determination of the elementary composition of the virus. Purified preparations of poliovirus obtained by the use of this method were inactivated with formaldehyde and were found to induce neutralizing antibodies in animals and man.
INTRODUCTION
The use of purified virus for the preparation of inactivated poliomyelitis vaccines and the demonstration of antibody response to these materials have been previously reported by Charney et al. (1957), Charney (1957)) and Hilleman et al. (1960a,b).
Several advantages are inherent in the use of such purified virus vaccine. Incorporation of virus antigen in precisely measured concentrations may be expected to yield a vaccine of controlled and reproducible potency. The removal of all serologically detectable monkey kidney antigen during purification “obviates the nagging theoretical question of whether nephrotoxicity and other reactions will develop eventually following multiple injections of poliomyelitis, respiratory, and other vaccines of monkey kidney origin in years to come” (Weihl et al., 1961).
The primary consideration in the successful preparation of such a vaccine is the development of a method for the preparation, without excessive loss, of virus sufficiently pure and concentrated to permit of precise quantification by physical methods. In a previous study (Mayer et al.; Schwerdt and Schaffer ; and Charney; 1957a), a modification of the Schwerdt and Schaffer procedure (Schwerdt and Schaffer, 1955) was described which employed an acid precipitation of the virus from partially purified concentrates. Further studies revealed the essential role of nucleic acid in this precipitation. This observation led to the development of a new procedure for virus purification involving the initial precipitation of virus at low pH in the presence of added nucleic acid. This procedure and the properties of the purified
virus obtained are described in this report.
MATERIALS AND METHODS
Solutions and Media
- PB, 7.0: Potassium phosphate buffer, 0.063 f&f, pH 7.0.
- PB, 3.5: Potassium phosphate buffer, 0.063 M, pH 3.5.
- SPB, 7.0: Potassium phosphate buffer, 0.063
- M, pH 7.0, containing 2% NaCI.SPB, 8.5: Potassium phosphate buffer, 0.063
- M, pH 8.5, containing 2% NaCl.
- NAc : Nucleic acid, commercial (Schwarz Laboratories, Mt. Vernon, New York) dissolved in NaOH to yield a 20% Solution, pH 7.0, and filtered.
- NAp : A polymerized fraction of nucleic acid was obtained from NAc by precipitation of a 0.1% solution of NAc in PB, 7.0 by addition of 1 iV HCl to pH 2.5 at 5”. The precipitate was redissolved by addition of dilute NaOH to pH 7.0 and filtered.
- Filter aid: Celite 503, Johns-Manville Corp., Philadelphia, Pennsylvania.
- Ribonuclease (RNase) : 3~ crystallized, Nutritional Biochemicals, Cleveland,
- Ohio. Dissolved in PB, 7.0 and sterilized by filtration.
- Deoxyribonuclease (DNase) : 1 X crystallized, Nutritional Biochemicals, Cleveland, Ohio. Dissolved in PB, 7.0 and sterilized by filtration.
Virus
Type 1 strain (Parker) (TC 52-146) obtained from Dr. David Bodian on 10/27/55. Type 2 strain MEFl and type 3 strain Saukett, routinely used for Salk poliomyelitis vaccine production.
TCF. The poliovirus tissue culture fluids used in these studies were obtained by the growth of types 1, 2, and 3 poliovirus on monolayers of monkey kidney cells. After harvesting, the fluids were filtered through Seitz-type clarifying and sterilizing pads. The virus-containing filtrate is designated TCF.”
EXPERIMENTAL FINDINGS
Orienting and Background Studies
“Earlier studies resulted in the development of a procedure for the purification of poliovirus involving the initial precipitation of virus at pH 4.5 in the presence of added methanol, followed by filtration with filter aid and elution of the filter cake with buffer. Further purification of the virus-rich eluate was effected by precipitation at pH 2.5 (Mayer et al.; Schwerdt and Schaffer; and Charney, 1957). Although initially reliable, the method was abandoned because precipitation of the virus at pH 2.5 was not uniformly successful. Failures were apparently correlated with a change of initial TCF filtration procedure from sintered glass (Corning) to Seitz-type pad filtration.
It was observed that in those instances in which acid precipitation of virus was successful, the final ultracentrifugal supernates exhibited a spectrophotometric adsorption spectrum suggestive of nucleic acid or nucleoprotein. The observation suggested that the acid precipitation of virus succeeded because of the presence of nucleic acid or nucleic acid degradation products. This possibility was strengthened by the finding that Seitz pad filtration, but not filtration through sintered glass, removed from TCF an acid-precipitable substance with an absorption spectrum similar to that of nucleic acid, thus offering an explanation for the failure of the acid precipitation step when applied to such fluids. This concept was confirmed by the demonstration that purified poliovirus concentrates were precipitated at pH 2.5 in the presence, but not in the absence, of added yeast nucleic acid.
Nucleic Acid Precipitation of Poliovirus
from TCF
The finding that nucleic acid serves as a
viral precipitant at low pH was applied directly to TCF. It was demonstrated that essentially complete precipitation of poliovirus was effected by addition of nucleic acid to TCF, followed by acidification to pH 2.5 with 1 N HCl at 5*; acidification of TCF in the absence of added nucleic acid was completely ineffective. Such precipitation was utilized as the first step in a new method for the concentration and purification of poliovirus. A number of variables affecting the precipitation were studied.
Precipitation of Virus by Nucleic Acid as a
Function of pH
NAc was added to a number of different
lots of type 1 (Parker) TCF to a level of 200 pg/ml. Aliquots were adjusted to various pH values with 1 N HCI at O-5”. The aliquots were centrifuged for 15 minutes at 4000 rpm (International, model PR2, 840 rotor) at 2”. The supernates were discarded, the precipitates taken up in one-tenth volume SPB, 7.0, further diluted tenfold (to original TCF volume) with PB, 7.0, and the recovered virus was assayed by CF. Excellent recoveries were obtained at pH 2.5 and 3.5. Below pH 2.5 recovery of virus was variable and was possibly related to destruction of viral antigen at the low pH. Recovery was also irregular at pH 4.0-4.5. Above pH 4.5 precipitation did not take place and no virus was recovered.
Stability of Virus-Nucleate Precipitate at
Low pH
In large-scale purification, for which this
procedure was designated, it is important that the precipitated virus remain uninjured during the time required for settling of the precipitate and separation of the supernate. To test this point, type 3 (Saukett) TCF containing 5 pg/ml of NAp was adjusted to pH 2.5 at 5*. At increasing intervals up to 24 hours, the bulk suspension was stirred, an aliquot removed, and the precipitate separated by centrifugation (2500 rpm; International centrifuge; 840 rotor) and dissolved in SPB, 7.0, to original volume. The samples were assayed for CF antigen and infectivity. Within the error of the assays used, complete stability was demonstrated under these conditions. A similar experiment using NAc showed both the CF and infectivity to be stable at pH 3.5 for at least 3 days.
The Poliovirus Purification Process
After delineation of the optimal conditions for precipitation of poliovirus from TCF with nucleic acid, procedures were developed for the further purification of the precipitated virus. The final method (Fig. 1) consisted in precipitation of the virus with nucleic acid at low pH; packing the settled precipitate by centrifugation; washing the packed precipitate with acid buffer; and solution of the washed precipitate in neutral buffer by addition of NaOH. Further removal of impurity was achieved by NaCl fractionation, the virus remaining in suspension at high ionic strength (Table 2). The purified viral suspension was pelleted by ultracentrifugation.
The ultracentrifuged pellets contained virus and, in the case of type 1 poliovirus, “C” component. The only other substantial impurity present in solutions of the pellets were split products of nucleic acid, with which the pellet and tube were wet. Almost all the impurities could be removed by washing the tube and pellet carefully with distilled water, but for complete removal, solution of the pellet and resedimentationwas required.
The ultracentrifuged virus pellets were
dissolved in SPB, 8.5, by incubation for 1
hour at 37”. A virus concentration of l-2
mg/ml resulted at this point. Over-all yields of the order of 50% have been obtained.
Purity of Final Virus Concentrates
The estimation of purity necessarily involves the use of some physical or chemical property of the virus for the determination of viral concentration. Ultracentrifugal sedimentation boundary analysis may be employed to determine virus concentration if the specific refractive increment of poliovirus is known. A value for this constant of 0.0018 has been proposed in the case of nucleoproteins including viruses (Englander and Epstein, 1957). Another convenient measure is the spectrophotometric absorption of virus concentrates free from, or corrected for, extraneous absorption. The use of such a measure requires a determination of the extinction coefficient of poliovirus, Schwerdt and Schaffer (1955) give a value for this constant E1%260 = 67. Using this value to measure the virus concentration in our best preparations, calculated virus nitrogen values (virus X 0.16) have routinely yielded nitrogen contents greater than the total nitrogen as determined directly; i.e., our preparations appeared to be more than 100% pure. Some uncertainty in the magnitude of the published value for the extinction coefficient of poliovirus thus seems to be indicated. Although the uncertainty indicated (unreadable) (approximately 22%), it is nevertheless troublesome when the calculation of virus purity is dependent upon this costant. It thus seemed necessary to redetermine the constant with as great a precision as possible.”
An analysis of the purity of the three
components (Parker, MEFl, and Saukett)
of a routine production lot is given in Table 4. The purified virus concentrates were prepared by the method shown in Fig. 2 and were subjected to two cycles of final ultracentrifugation. The final preparations were essentially pure (98.5-103%)) after correction for the “C” component (of viral origin) in the type 1 preparation. (“C” component has been found in all but one of the type 1 virus lots purifed. Significant quantities of “C” component have not been found in freshly purified type 2 (MEFl) or type 3 (Saukett) preparations.) Monkey kidney antigen, as measured by CF (MKCF), was undetectable in all the purified preparations when tested at a level of approximately 150 pg/ml virus. This represents a reduction in MKCF, where detectable in TCF, of >210-fold (type 1) and >850-fold (type 3). Similar results with respect to yield and purity have been obtained for all the routine production lots purified by this procedure, resulting in the preparation of gram quantities of essentially pure poliovirus.
Further purity determinations of a number of routine preparations have shown that type 2 (MEFl) and type 3 (Saukett) preparations have not differed significantly from 100% purity when doubly ultracentrifuged. For type 1 (Parker) purified concentrates, where spectrophotometry was shown to yield only approximate estimates, purity was more rigorously determined by ultracentrifugal sedimentation boundary analysis. Purity analyses for six routine preparations are shown in Table 5. The difference in viral concentration as determined by spectrophotometry and sedimentation analysis is also shown in Table 5.
The purities of the six preparations analyzed averaged 98.2%. The range of values (80-112%) reflects the precision with which such estimates can be made. With respect to sedimentation analysis, spectrophotometry overestimated the virus concentration in these preparations by an average of 22%.
With this demonstration of satisfactory
yield and excellent purity, the original objectives of this study, namely the development of a procedure for the preparation of purified poliovirus for vaccine production in satisfactory yield and with a purity sufficient to permit of determining concentration by relatively precise physical methods, were judged to have been attained.”
“Because of the difficulties associated with the weighing and handling of possibly infectious dry preparations in a nonviral area by the analytical group, the elementary analytical composition was determined using formaldehyde-inactivated poliomyelitis antigen. The virus at aproximately 1400 rg/ml was incubated with 92.5 pg/ml .HCHO (1:4000 formalin) at 37” for 8 days. The inactivated virus was pelleted by ultracentrifugation, and the pellets and tubes were washed twice with distilled water. The pellets were dispersed with 10 ml of distilled water, whereupon the material crystallized. The crystalline slurry was lyophilized, and the lyophilized product was dried for 3 days over P205 under a vacuum of 0.2 mm Hg at room temperature. Sedimentation boundary analysis of the formaldehyde-inactivated virus, prior to lyophilization, showed no change in sedimentation constant, diffusion constant, or molecular weight, a result indicating that the amount of formaldehyde combined could not affect the analytical results. The elementary analytical results for the antigen, therefore, can be assumed to be correct for the virus as well.
The large residue found on ignition requires interpretation, since a true ash of this dimension seemed impossible. Since ignition was conducted at a temperature (850”) that insured the volatility of P205 , the only explanation appeared to be fixation of a portion of the P by cation (Na) bound to the neutralized virus, NaP03 forming the ash. A residue of 9.06% NaPOa implies an Na content of 2.16%. To confirm this interpretation, a “synthetic virus” preparation, containing nucleic acid and protein in approximately the ratio found in poliovirus, was made by combining 30 parts of yeast nucleic acid with 70 parts of ash-free bovine serum albumin. The mixture required approximately 2.2% of Na (as NaOH) to bring a solution of the preparation to pH 7.01. The lyophilized, neutralized preparation, which exhibited an infrared spectrum virtually indistinguishable from that of lyophilized polio antigen, yielded an ash of 9.75%.
Correction of the elementary analysis to
allow for Na content (factor: 1.021) yields
the figures in Table 6, column b.
A final correction may be applied to compensate for the formation of “C” particles; the inactive antigen was found to contain 10% of this component by sedimentation boundary analysis. At the same time, evidence of the release of nucleic acid during incubation was shown by distortion of the ultraviolet spectrum during inactivation. These data indicate that “C” component originated during incubation at 37” for 8 days by breakdown of virus nucleoprotein into nucleic acid and nucleate-free protein. Since “C” component is believed to contain little or no nucleic acid (Le Bouvier et al., 1957), it appears that the figures in Table 6, column b should be corrected for the presence of approximately 10% of protein in order to arrive at the true elementary composition of isoelectric poliovirus. If “C” component does contain a residuum of nucleic acid, the small corrections for its presence will be further lessened. Assuming a mean elementary composition for protein of: C = 50%, N = IS%, and H = 7.0%, the corrected composition is given in Table 6, column c.”
(The various considerations involved in arriving at the best estimate of the elementary composition of poliovirus may seem unduly numerous, but the total corrections are small and are given here largely for the purpose of explication. The constants for poliovirus presented in this report refer to purified materials exhibiting physical homogeneity. This does not preclude the possible presence, however, of small and unknown amounts of other viruses of similar physical and chemical composition which might have been derived from the monkey kidney cell tissue culture fluids from which the poliovirus was purified.)
doi.org/10.1016/0042-6822(61)90358-0
From Charney’s first paper, it is clear that the so-called purification is based on estimates rather than EM imagery showcasing nothing but homogeneous particles. He used both ultracentrifugal sedimentation boundary analysis and spectrophotometric absorption to determine “virus” concentration, however they both require specific knowledge about the “virus” in order to be accurate. Without having purified/isolated “virus,” to begin with in order to determine this information, the values used are essentially guesstimates. This was proven by the redetermination of the extinction coefficient they originally used from Schwerdt which Charney decided gave too much uncertainty. Without accurate numbers, purity could not be determined.
The “virus” as usual comes from unpurifued monkey tissue cultures instead of directly from humans. The tissue culture fluid, which had already been subjected to unknown chemical additives, is further subjected by the addition (not subtraction as should be the case with purification/isolation) of multiple potassium phosphate buffers, nucleic acids (from yeast), RNase, DNase, Celite 503 filter aid, etc. Charney admits to impurities such as the “C” component (assumed to be part of the “virus”) within the samples and actually states that their calculations do not account for small and unknown amounts of other “viruses” of similar physical and chemical composition which might have been derived from the monkey kidney cell tissue culture fluids from which the poliovirus was “purified.” In other words, this is not evidence of purification/isolation of any Polio “virus.”
This second study from Charney a few months later in September 1961 details how his method could be used to obtain a “purified” Polio vaccine. The highlights from this paper are proof that the Salk vaccine, which had been on the market since 1955, was nothing but unpurified toxins:
Development of a Purified Poliomyelitis Virus Vaccine
Salk poliomyelitis vaccine, as ordinarily prepared, exhibits marked variation in antigenic potency from lot to lot. In spite of high over-all efficacy, the vaccine does not protect all individuals against the disease, even after the fourth dose.
During the past several years, our laboratories have sought to develop a purified poliovaccine which would exhibit uniform high potency and which would overcome the deficit in protective capacity of the ordinary m preparation. This goal has been achieved. In the process, the vaccine has been freed of all serologically detectable monkey kidney substance and of essentially all other nonviral contaminating substances present in the ordinary crude commercial Salk vaccine.
The present report describes the method for preparing and testing the purified vaccine. Preliminary studies relating to the development and evaluation of the purified vaccine have been presented elsewhere.
Improvements to be desired in commercial Salk poliomyelitis vaccine are the elimination of lot-to-lot variations in potency, increase in mean vaccine potency, and elimination of foreign antigens, particularly host (monkey kidney) antigen. These objectives have been achieved by the inactivation of purified, concentrated poliomyelitis virus to give a vaccine containing standard optimal quantities of poliomyelitis antigen which are essentially free from nonviral antigens. The relatively avirulent Parker type I poliovirus was substituted for the highly neurovirulent type I Mahoney strain. Removal of the monkey kidney antigen minimized the hazard of inducing allergic or auto-immune reactions. The preparation and testing of this vaccine are described.
Results
Vaccine Preparation Method.—The method for preparing and testing the vaccine conforms in all respects to the standards for poliomyelitis vaccine as outlined in the U.S. Public Health Service Regulations for Biological Products. Figure 1 outlines the general method for preparing the purified vaccine. The principal difference from the ordinary Salk vaccine lies in the fact that the viral antigen in the vaccine is essentially pure; the amount of viral antigen put into the vaccine is precisely measured; and the highly neurovirulent type I Mahoney virus of the ordinary vaccine is replaced by the relatively avirulent Parker strains in keeping with the objectives of the Technical Committee on Poliomyelitis Vaccine. The type II and III virus strains are the same as in the ordinary vaccine.
Virus Purification.—The virus purification procedure stems from the early pioneering experiments of Schwerdt and Schaffer who purified poliovirus by a combined methanol fractionation and differential centrifugation method. This was followed by the work of Charney et al. who devised an efficient method for poliovirus purification using initial viral concentration by precipitation with nucleic acid and combined physical and chemical separation.
The method for virus purification is outlined in Figure 2. After filtration through a Seitz pad, the virus was precipitated from the crude infected tissue culture fluid using nucleic acid at low pH. The virus nucleate precipitate was washed with buffer solution, dissolved by neutralization, and treated, with nucleases to degrade cell-derived and reagent nucleic acid. Impurities were removed by precipitation with 7.5% sodium chloride at pH 4.5 and the virus was recovered from the salt solution by ultracentrifugation. The final concentrate was clarified by centrifugation or by filtration.
Standardization of the amount of each viral antigen in the final vaccine depended on precise quantitative assay of the viral concentrates from which the noninfective immunizing antigens were prepared. Such assays were performed on the clarified viral concentrates, just prior to inactivation, by spectrophotometric determination of optical density, by ultracentrifugal boundary analysis, and by assay for total nitrogen. The best measure of purity and virus content was by boundary analysis based on the specific refractive increment for poliovirus (0.00174). Spectrophotometry was useful for types II and III poliovirus, but presence of nonviral absorption rendered this determination only approximate for type I.
The poliovirus concentrates used to prepare the vaccine usually were essentially pure. “C” component was commonly present in the type I virus preparations. This virus-associated entity sedimented more slowly than did the poliovirus and is thought to comprise the protein coat of the polio-virus particle devoid of nucleic acid. Since this entity was virus-related and since its capacity to stimulate neutralizing antibody was uncertain, it was counted neither as impurity nor as viral antigen.
In certain preparations, nondialyzable, nonviral material was present in trace amount. This presumably was of host cell origin and was considered undesirable insofar as the vaccine was concerned. In no case, however, did the concentrates contain serologically detectable monkey kidney complement fixation (CF) antigen, even when tested in high concentration. A small amount of nucleic acid was sometimes present in viral concentrates which had not been subjected to a double cycle of high speed centrifugation. This was inert material antigenically and was not counted as a significant impurity of the final product.
Calculations to appraise the purity of the individual components of 4 trivalent lots of purified poliovaccine are presented in Table 1. The total nitrogen was determined according to Johnson. The virus and “C” components were measured by sedimentation analysis and reduced to nitrogen content by the usual factor of 0.16. Extraneous nucleic acid, when present, was measured by spectrophotometry and was calculated to nitrogen by the factor 0.16.
Virus purity was computed by the formula:
The calculated purity for the lots shown in Table 1 ranged from 81-131%, with an average value of 103%. Data relating to the degree of purification achieved in the process of purification from the crude infected tissue culture fluids of the individual components of lot 11 trivalent vaccine (see Table 1) are presented in Table 2. The data are summarized in terms of reduction of extraneous protein impurity and in terms of reduction in content of monkey kidney CF antigen. The amount of nonviral protein impurity in the crude infected tissue culture fluids ranged from 98.3% to 99.2% of the total protein content of the fluids. By contrast, only 13.5% of the total protein of the Parker type I purified concentrate was nonviral and the type II and III concentrates were free of such extraneous protein.
The amount of monkey kidney CF antigen per y of poliovirus was reduced more than 60 and 300-fold for types I and II, respectively, in the concentrates. The fold-reduction for the type III component cannot be given since the level of monkey kidney CF antigen in the original crude fluid was below the sensitivity of the CF test. The purified concentrate, however, showed no detectable CF antigen even though the virus was concentrated 129 times.
The degree of purity achieved may be appreciated in the light of the fact that the measurable properties of the virus concentrates routinely used for purified poliovaccine production do not differ
significantly from the classical preparations of purified poliovirus described by Schwerdt and Schaffer. The ultraviolet absorption spectra for 3 typical purified poliovirus concentrates are shown in Figure 3. These are characteristic of poliovirus. The ultracentrifugal boundary diagrams of the 18 components of 6 trivalent vaccine lots are shown in Figure 4. The only boundaries seen in the types II and III concentrates are those of the virus. Type I concentrates exhibit “C” particle as well as virus.”
Comment
The ideal objective of a standardized poliovirus vaccine of uniform high potency has been achieved through the viral purification and concentration procedure. Substitution of the relatively avirulent Parker type I poliovirus for the highly neurovirulent type I Mahoney strain adds a “margin of safety” to the purified vaccine. Removal of the monkey kidney antigen minimizes the potential for induction of allergic or autoimmune reactions which might conceivably develop following repeated injections of vaccines of monkey kidney origin.”
doi:10.1001/jama.1961.03040350001001
It should be clear after reading Charney’s second study that the Salk Polio vaccine was nothing short of an impure contaminated mess which could (and did) potentially cause severe allergic reactions and autoimmune disorders. Charney claims his method offered an “essentially pure” vaccine yet he admittedly ignored impurities such as the “C” component as well as extraneous nucleic acids said to be of monkey origin. Charney, like all virologists, loved to play with semantics which is why he allowed for an “essentially pure” category even though this implies it is not pure. Perhaps this is why the Salk vaccine, even with Charney’s supposed improvements, went unused in the US from 1963 until it was retooled in 1999?
This didn’t stop Charney from applying for a patent on his purification method in 1964. Some highlights from his application provide even more details as to the methods of his madness:
Process for purifying viral substances and composition
Claims
1. A PROCESS FOR PURIFYING AND CONCENTRATING POLIOMYELITIS VIRAL SUBSTANCES FROM POLIOMYELITIS VIRAL SUBSTANCE-CONTAINING FLUIDS, COMPRISING ADDING NUCLEIC ACID TO THE POLIOMYELITIS VIRAL SUBSTANCE-CONTAINING FLUID AND ADJUSTING THE PH TO BETWEEN 2-4 AND RECOVERING THE PRECIPITATED POLIOMYELITIS VIRAL SUBSTANCE.
“This invention relates to an improved method for concentrating and purifying viral substances. More particularly it relates to a process of separating, in highly concentrated and purified form, viral substances from viral substance-containing fluids such as tissue culture fluids or Vaccines made by the elimination of the infective property of tissue culture fluids by reaction with formaldehyde, long incubation, etc., and further relates to the use of pure concentrated viral substances in the preparation of vaccines, and the vaccines thus produced.
The preparation of vaccines for use in preventing diseases of viral origin, particularly poliomyelitis, has recently been simplified by growing the causative virus in tissue culture. The tissue culture media may be of several types, for example in growing poliomyelitis in monkey kidney tissue culture, the monkey kidneys are either minced or minced and trypsinized. The work described herein was done on minced and trypsinized monkey tissue culture fluids containing poliomyelitis virus (hereinafter referred to as poliovirus) but would be equally applicable to the minced tissue culture fluid with an even greater increase in the purification factor achieved, or to fluids or filtrates obtained from other tissue cultures.
At the end of the poliovirus growth period, tissue culture fluids are extremely dilute solutions of the desired viral substances contaminated with large amounts of impurities, many of them undesirable.
While methods are known which are used to concentrate and purify viral substances from tissue culture filtrates, these require that a large number of procedural steps be employed with the attendant loss in yields and opportunity for contamination. In addition they are tedious and time consuming. For example, the method described by Schwerdt & Schaffer, Annals of the New York Academy of Sciences, volume 61: article 4, page 740 (1955), by which poliovirus was obtained in a pure state, prior to applicants invention, involved (1) precipitation of the virus in the presence of methanol, filtration of the virus so precipitated with the aid of a carrier (such as Celite), and elution of the filter-cake, done repeatedly; (2) repeated emulsification with butanol to remove protein; (3) digestion with enzymes; (4) two cycles of ultracentrifugation; (5) final ultracentrifugation in a sucrose solution of graded density; and (6) removal of the sugar.
It can readily be seen that such complicated techniques applied to large quantities of dangerously infective virus fluids are not attractive from the point of view of commercial manufacture and that a method which simply and quickly results in an essentially pure virus product would be of great importance.
The disadvantages and hazards of the known methods for purifying and concentrating viral substances are completely overcome by the method of my invention which provides a means for obtaining pure viral substance quickly and simply and in substantially quantitative yields from impure virus-containing fluids or vaccine. The process of my invention shown in FIG. 1 of the accompanying drawings, is based upon a new principle, namely that viral substances can be precipitated quantitatively and in highly purified form from dilute or concentrated impure virus containing solutions by adding nucleic acid and adjusting the solution to a pH between about 2-4.“
“The advantages of the use of nucleic acid to eifect concentration of viral substances from tissue culture fluid are manifold: (1) very small quantities are required, 50-200 nag/liter of crude commercial product or 0.5-2 mg./liter of purified product are suflicient to effect the precipitation of the viral substance, higher concentrations can be employed, if desired, although not usually necessary; (2) the nucleic acid is easily obtainable and inexpensive, especially if yeast nucleic acid is used; (3) the precipitate of nucleic acid and virus is readily soluble in neutral aqueous solutions; (4) successive precipitations can be carried out without loss of viral matter; (5) and the nucleic acid can be separated from the virus material by digestion with a nuclease such as ribonuclease or a mixture of ribonuclease and deoxyribonuclease. The split products of the nucleic acid digestion then can be eliminated by dialysis, ultracentrifugation or reprecipitation of viral material by the addition of an alcohol, for example ethanol or methanol, the digestion products remaining in the supernatant.”
“In general, the product obtained at this point represents substantially pure poliovirus, the poliovirus being 80- 100% pure. The major impurity, if any, at this point, is the material known to the art as C component which is related to the poliovirus, and is, in fact, the outer protein envelope of the virus particle which lacks the central core of nucleic acid. Inactivation of the virus thus obtained by methods known to the art, will result in substantially pure poliovirus antigen. However, if the process is performed using an imperfectly filtered tissue culture fluid or without due care, the product obtained will not be substantially pure. Nevertheless, even under these conditions, the product will contain poliovirus as a major component with respect to the total protein in the concentrate, and inactivation of this product by methods known to the art will yield a vaccine containing poliovirus antigen as the major portion of the total protein in the vaccine. The vaccine so obtained is fully effective for immunization against poliomyelitis disease and could be sold as a marketable product.
The preparation of purified viral material by the method of my invention is of considerable commercial importance because for the first time it is now possible to accomplish its concentration and purification in considerably less time than was needed when employing known procedures, and the virus is obtained in substantially quantitative yields. The purified and concentrated virus which is obtained by the process of my invention if of commercial importance because it can be converted to a purified vaccine largely or wholly freed from undesirable matter.”
https://patents.google.com/patent/US3147185A/en
In Summary:
- According to Charney, the precipitation of polio by nucleic acid at low pH represented a new, highly satisfactory method for the concentration of “virus” from large volumes of tissue culture filtrate
- Virtually (i.e. nearly; almost) pure preparations obtained permitted the redetermination of the extinction coefficient of polio (E&i = 81.6) and the determination of the specific refractive increment of the “virus” (An = 0.00174)
- The removal of all serologically detectable monkey kidney antigen during purification obviates the nagging theoretical question of whether nephrotoxicity and other reactions will develop eventually following multiple injections of poliomyelitis, respiratory, and other vaccines of monkey kidney origin in years to come
- In a previous study, a modification of the Schwerdt and Schaffer procedure was described which employed an acid precipitation of the “virus” from partially purified concentrates
- Further studies revealed the essential role of nucleic acid in this precipitation which led to the development of a new procedure for “virus” purification involving the initial precipitation of “virus” at low pH in the presence of added nucleic acid
- The poliovirus tissue culture fluids used in these studies were obtained by the growth of types 1, 2, and 3 poliovirus on monolayers of monkey kidney cells
- Previous purification methods by Schwerdt, Shaffer, and Charney were abandoned because precipitation of the “virus” at pH 2.5 was not uniformly successful
- Failures were apparently correlated with a change of initial TCF filtration procedure from sintered glass (Corning) to Seitz-type pad filtration
- It was observed in instances in which acid precipitation of “virus” was successful, the final ultracentrifugal supernates exhibited a spectrophotometric adsorption spectrum suggestive of nucleic acid or nucleoprotein
- The observation suggested that the acid precipitation of “virus” succeeded because of the presence of nucleic acid or nucleic acid degradation products
- Charney states that this concept was confirmed by the demonstration that purified poliovirus concentrates were precipitated at pH 2.5 in the presence, but not in the absence, of added yeast nucleic acid
- The finding that nucleic acid serves as a “viral” precipitant at low pH was applied directly to TCF which demonstrated that essentially complete precipitation of poliovirus was effected by addition of nucleic acid to TCF
- Excellent recoveries were obtained at pH 2.5 and 3.5 yet below pH 2.5 recovery of “virus” was variable and was possibly related to destruction of “viral” antigen at the low pH
- Recovery was also irregular at pH 4.0-4.5 and above pH 4.5 precipitation did not take place and no “virus” was recovered
- Charney states it is important that the precipitated “virus” remain uninjured during the time required for settling of the precipitate and separation of the supernate
- The final method consisted of:
- Precipitation of the “virus” with nucleic acid at low pH
- Packing the settled precipitate by centrifugation
- Washing the packed precipitate with acid buffer
- Solution of the washed precipitate in neutral buffer by addition of NaOH
- The ultracentrifuged pellets contained “virus” and, in the case of type 1 poliovirus, “C” component
- The only other substantial impurity present in solutions of the pellets were split products of nucleic acid, with which the pellet and tube were wet
- Almost all the impurities could be removed by washing the tube and pellet carefully with distilled water
- The estimation of purity involves the use of some physical or chemical property of the “virus” for the determination of “viral” concentration
- Ultracentrifugal sedimentation boundary analysis may be employed to determine “virus” concentration if the specific refractive increment of poliovirus is known
- A value for this constant of 0.0018 has been proposed in the case of nucleoproteins including “viruses” by Englander and Epstein in 1957
- Another convenient measure is the spectrophotometric absorption of “virus” concentrates free from, or corrected for, extraneous absorption
- The use of such a measure requires a determination of the extinction coefficient (refers to several different measures of the absorption of light in a medium) of poliovirus, Schwerdt and Schaffer in 1955 gave a value for this constant E1%260 = 67
- Some uncertainty in the magnitude of the published value for the extinction coefficient of poliovirus was indicated
- The uncertainty indicated (approximately 22%) was troublesome when the calculation of “virus” purity is dependent upon this costant
- He decided to redetermine the constant with as great a precision as possible
- The final preparations were essentially pure (98.5-103%) after correction for the “C” component (assumed to be of “viral” origin) in the type 1 preparation
- “C” component has been found in all but one of the type 1 “virus” lots purifed
- Monkey kidney antigen, as measured by (indirect) Compliment Fixation (MKCF), was undetectable in all the purified preparations when tested at a level of approximately 150 pg/ml “virus”
- This represents a reduction in MKCF, where detectable in TCF, of >210-fold (type 1) and >850-fold (type 3)
- Similar results with respect to yield and purity have been obtained for all the routine production lots purified by this procedure, resulting in the preparation of gram quantities of essentially pure poliovirus
- Spectrophotometry was shown to yield only approximate estimates
- The purities of the six preparations analyzed averaged 98.2% based on their estimates
- Charney believed the range of values (80-112%) reflects the precision with which such estimates can be made
- With respect to sedimentation analysis, spectrophotometry overestimated the “virus” concentration in these preparations by an average of 22%
- The objective of the study was the development of a procedure for the preparation of purified poliovirus for vaccine production in satisfactory yield and with a purity sufficient to permit of determining concentration by relatively precise physical methods (in other words, not completely pure determined by not completely precise means)
- Because of the difficulties associated with the weighing and handling of possibly infectious dry preparations in a nonviral area by the analytical group, the elementary analytical composition was determined using formaldehyde-inactivated poliomyelitis antigen (in other words, their “purified virus” prep was too dangerous so inactivated “virus” antigen obtained in another way was used)
- Charney claimed that the elementary analytical results for the antigen could be assumed to be correct for the “virus” as well
- Large residue found on ignition required interpretation, since a true ash of this dimension seemed impossible
- To confirm their interpretation, a “synthetic virus” preparation, containing nucleic acid and protein in approximately the ratio found in poliovirus, was made by combining 30 parts of yeast nucleic acid with 70 parts of ash-free bovine serum albumin
- The mixture required approximately 2.2% of Na (as NaOH) to bring a solution of the preparation to pH 7.01
- The lyophilized, neutralized preparation, which exhibited an infrared spectrum virtually indistinguishable from that of lyophilized polio antigen, yielded an ash of 9.75%
- Their data indicated that “C” component originated during incubation at 37” for 8 days by breakdown of “virus” nucleoprotein into nucleic acid and nucleate-free protein
- “C” component was believed to contain little or no nucleic acid
- However, if “C” component does contain a residuum of nucleic acid, the small corrections for its presence would be further lessened (in other words, they had no idea what this “C” component was)
- In a final blow to the validity of the whole study (included in the side notes of all places) was the revelation that their estimates does not preclude (i.e. prevent) the possible presence of small and unknown amounts of other “viruses” of similar physical and chemical composition which might have been derived from the monkey kidney cell tissue culture fluids from which the poliovirus was “purified”
- Salk poliomyelitis vaccine, as ordinarily prepared, exhibits marked variation in antigenic potency from lot to lot
- The vaccine did not protect all individuals against the disease, even after the fourth dose
- During the past several years, Charney’s labs sought to develop a purified poliovaccine which would exhibit uniform high potency and which would overcome the deficit in protective capacity of the ordinary preparation
- The vaccine has been freed of all serologically detectable monkey kidney substance and of essentially all (i.e not all) other nonviral contaminating substances present in the ordinary crude commercial Salk vaccine
- Charney claims his objective was achieved to give a vaccine containing standard optimal quantities of poliomyelitis antigen which were essentially free (there’s that phrase again) from “nonviral” antigens
- Removal of the monkey kidney antigen minimized the hazard of inducing allergic or auto-immune reactions
- According to Charney, the principal differences from the ordinary Salk vaccine is that:
- The “viral” antigen in the vaccine is essentially pure
- The amount of “viral” antigen put into the vaccine is precisely measured
- The highly neurovirulent type I Mahoney “virus” of the ordinary vaccine is replaced by the relatively avirulent Parker strains
- The “virus” purification procedure stems from the early experiments of Schwerdt and Schaffer and was revised later by Charney
- The method consisted of these steps:
- After filtration through a Seitz pad, the “virus” was precipitated from the crude infected tissue culture fluid using nucleic acid at low pH.
- The “virus” nucleate precipitate was washed with buffer solution
- Dissolved by neutralization
- Treated with nucleases to degrade cell-derived and reagent nucleic acid
- Impurities were removed by precipitation with 7.5% sodium chloride at pH 4.5 and the “virus” was recovered from the salt solution by ultracentrifugation
- The final concentrate was clarified by centrifugation or by filtration
- The best measure of purity and “virus” content was by boundary analysis based on the specific refractive increment for poliovirus (0.00174) (without having purified/isolated Polio first, how was this number derived in order to measure purity?)
- Spectrophotometry was useful for types II and III poliovirus, but presence of “nonviral” absorption rendered this determination only approximate for type I
- The poliovirus concentrates used to prepare the vaccine usually were essentially pure (they love to use this oxymoron)
- “C” component (the assumed “viral” fragment) was commonly present in the type I “virus” preparations
- This “virus-associated” impurity was thought to comprise the protein coat of the Polio particle devoid of nucleic acid
- Since this entity was “virus-related” and since its capacity to stimulate neutralizing antibody was uncertain, it was counted neither as impurity nor as “viral” antigen
- In certain preparations, nondialyzable, nonviral material was present in trace amount and this presumably was of host cell origin and was considered undesirable insofar as the vaccine was concerned
- A small amount of nucleic acid was sometimes present in “viral” concentrates which had not been subjected to a double cycle of high speed centrifugation
- This was considered inert material antigenically and was not counted as a significant impurity of the final product
- Extraneous nucleic acid, when present, was measured by spectrophotometry and was calculated to nitrogen by the factor 0.16
- The calculated purity for the lots shown in Table 1 ranged from 81-131%, with an average value of 103%
- The data was summarized in terms of reduction of extraneous protein impurity and in terms of reduction in content of monkey kidney CF antigen (reduction does not equal elimination)
- The amount of “nonviral” protein impurity in the crude infected tissue culture fluids ranged from 98.3% to 99.2% of the total protein content of the fluids
- By contrast, only 13.5% of the total protein of the Parker type I purified concentrate was “nonviral” and the type II and III concentrates were free of such extraneous protein
- The amount of monkey kidney CF antigen per y of poliovirus was reduced (not eliminated) more than 60 and 300-fold for types I and II
- Charney concludes that substitution of the relatively avirulent Parker type I poliovirus for the highly neurovirulent type I Mahoney strain adds a “margin of safety” (his quote marks, not mine) to the purified vaccine
- Removal of the monkey kidney antigen minimized the potential for induction of allergic or autoimmune reactions which might conceivably develop following repeated injections of vaccines of monkey kidney origin
- Charney describes his invention as a process of separating, in highly concentrated and purified form, “viral” substances from “viral substance-containing” fluids such as tissue culture fluids or vaccines (notice it does not claim purification/isolation of samples directly from humans)
- The work described in his patent was done on minced and trypsinized monkey tissue culture fluids assumed to contain poliomyelitis “virus”
- Charney admits that at the end of the poliovirus growth period, tissue culture fluids are extremely dilute solutions of the desired “viral” substances contaminated with large amounts of impurities, many of them undesirable
- He states that while methods are known which are used to concentrate and purify “viral” substances from tissue culture filtrates, these require that a large number of procedural steps be employed with the attendant loss in yields and opportunity for contamination
- Charney desires a method which simply and quickly results in an essentially pure (what is with that phrase?!?!) “virus” product
- He claims that the disadvantages and hazards of the known methods for purifying and concentrating “viral” substances are completely overcome by his invention which provided a means for obtaining pure “viral” substance quickly and simply and in substantially quantitative yields from impure “virus-containing” fluids or vaccine
- His method is based upon a new principle, namely that “viral” substances can be precipitated quantitatively and in highly purified form from dilute or concentrated impure “virus” containing solutions by adding nucleic acid and adjusting the solution to a pH between about 2-4
- An advantage is that the nucleic acid is easily obtainable and inexpensive, especially if yeast nucleic acid is used
- Using his method, in general, the product obtained represents substantially pure (a new descriptor!…yet it still doesn’t mean complete purification/isolation) poliovirus, being 80-100% pure
- The major impurity, if any, is the material known as “C” component which is assumed to be of “viral” origin
- However, if the process is performed using an imperfectly filtered tissue culture fluid or without due care, the product obtained will not be substantially pure
- Charney makes the caveat that even under these conditions, the product will contain poliovirus as a major component with respect to the total protein in the concentrate, and inactivation of this product will yield a vaccine containing poliovirus antigen as the major portion of the total protein in the vaccine
- Charney claims the purified and concentrated “virus” which is obtained by the process of his invention can be converted to a purified vaccine largely or wholly freed from undesirable matter
Partially purified, essentially purified, substantially purified, highly purified, relatively purified, virtually purified…all of these phrases fail the definition of purification which means to make free from anything that debases, pollutes, adulterates, or contaminates; to free from foreign, extraneous, or objectionable elements. It does not mean to free it from some or most of these things, it means to be freed from all of them. It is clear virology can not grasp the most basic of definitions.
When Charney discussed purification, he was doing so in regards to breaking down the already impure tissue culture fluids by adding nucleic acids and other chemicals in an attempt to dissolve “nonviral” elements. By his own methods and crude calculations, which did not account for potential “viruses” or impurities of the same size and chemical composition of the assumed Polio “virus,” Charney was unable to achieve complete purification/isolation of the “virus.” Even if he was able to do so, he never took EM photographs of these purified “virus” particles nor did he attempt to prove it was pathogenic. He would not have been able to prove that these particles were ever inside a human as nothing was ever purified/isolated directly from a sick human but came from monkey kidney tissue cultures instead. Everything Charney did was after the period where purification/isolation of a “virus” would have been needed to be done, i.e. the very first step in the discovery process, not afterwards when developing a vaccine.
What Charney did prove, unintentionally, is that the Salk vaccine was a toxic concoction that was rushed to be tested on the populace in an impure state. He also unintentionally proved that the claims of purification/isolation credited to Loring and Schwerdt were untrue. If a purified “virus” needed further purification/characterization years later by “improved” methods, it was never properly purified/isolated to begin with. Unfortunately for Charney, his methods must not have been highly regarded nor accepted as his contributions to the supposed purification/isolation of Polio amounts to one blurb in an obscure book.
It should be clear by now that there is no evidence of a Polio “virus” taken directly from a sick human which was then purified/isolated and proven pathogenic. What we do have is a history of horrors from the grotesque brain drilling experiments to the sickening emulsified brain/spinal goo that was injected directly into helpless animals. We have disgusting tissue culture experimemts which combined this toxic soup with various infant and monkey organs. We have a mixture of these disturbing practices put together from the brain/spine of a mouse, the emulsified CNS of a monkey, and the feces of a paralyzed patient combined into a monkey kidney tissue culture subjected to formaldehyde/sodium bisulfite as well as other unknown ingredients which was then injected directly into healthy people. We have evidence of inhumane and criminal fraud which has taken place for well over a hundred years now.
What we do not have is evidence of a properly purified/isolated “virus.”
ViroLIEgy 
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